The BACH-label was used in the original form without any purification after its purchase. The presence of an isomeric byproduct was evident when the chromatography of the native label was performed. Two peaks (tR: 16.8 min and 24 min, C18; 2 min and 6 min, HILIC) with the same m/z at 372 were present when chromatographically examined (data not shown). An example of this fact can be seen later in Fig.11 with the disaccharides, where the unreacted BACH shows two peaks. Due to this fact, we obtained after derivatization of saccharides a main product with a byproduct of the same m/z (Fig. 9). When necessary, the byproduct can be easily purified away with the excess BACH-label. Detailed structure of this byproduct is under investigation. Our further measurements concentrated only on the main product. As the BACH-tag eluted on the reversed phase between 16- 17 min with other sugars such as Man and Glc, a simultaneous clean-up of this label with the method on the C18 would not be feasible. However, when applied to HILIC-phase, the BACH-reagent eluted earlier than all the sugars and could be separated away when desired. As 2- Aminobenzamid label eluted before the mixture of monosaccharides on both stationary phases RP (6 min.) and HILIC (1.5 min.), a clean-up of the 2AB-label by this methodology would be very straightforward by both chromatographic methods.

66 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

derivatives provided a clear baseline resolution.

The BACH-derivatives interacted with the C18-phase ~8-15 min longer than the respective 2-AB-derivatives. The elution order of the sugars on the C-18-phase remained similar except for the triplet of the sugars galactose, glucose and mannose (Gal-2-AB, Glc-2AB, Man-2AB and Gal-BACH, Man-BACH, Glc-BACH). Galactose and mannose separation was achieved with both labels under the named conditions on the RP (Fig. 7), whereas the BACH-

Glucose is rarely present in glycoproteins. It can however originate from hydrolysis of contaminating polysaccharides from the environment. This problem was often encountered in reversed phase chromatography of monosaccharides (Toomre & Varki, 1994; Kwon & Kim, 1993) where glucose (at least partially) overlaps with the peak of galactose. Therefore,

In our experiments on C18 and HILIC-phase, 2-AB-derivatized glucose and galactose eluted very close to each other (Fig. 7 and Fig. 8, respectively). Through BACH-derivatization, on the other hand, glucose and galactose could have been separated on C18 (Fig. 7). However,

A complete separation of glucose from galactose and mannose was reached only after the application of BACH-sugars to the HILIC-phase as shown in Fig. 9. However, the monosaccharides galactose and mannose could have been separated from each other only by means of RP chromatography (Fig.7). In this regard, the two phases (RP and HILIC) provided complementary features and their interchange may help to solve some analytical obstacles.

The BACH-label was used in the original form without any purification after its purchase. The presence of an isomeric byproduct was evident when the chromatography of the native label was performed. Two peaks (tR: 16.8 min and 24 min, C18; 2 min and 6 min, HILIC) with the same m/z at 372 were present when chromatographically examined (data not shown). An example of this fact can be seen later in Fig.11 with the disaccharides, where the unreacted BACH shows two peaks. Due to this fact, we obtained after derivatization of saccharides a main product with a byproduct of the same m/z (Fig. 9). When necessary, the

glucose contamination in carbohydrate samples should be addressed and examined.

the separation of glucose from mannose was still insufficient with BACH-labeling.

**Figure 8.** Hydrophilic interaction LC/MS of 2-AB-derivatized monosaccharides

The peaks of the investigated monosaccharides on the HILIC phase show broadening or splitting (Fig. 9). This is due to mutarotation of the sugars and consequently the presence of its anomers. The broadening is more evident with sugars, which mutarotate fast e.g. glucose and mannose. This phenomenon was observed with small reducing sugars on the HILICphase already in the past (Churms, 1996; Moriyasu *et al.*, 1984).

**Figure 9.** Extracted ion chromatograms (EIC) of BACH-derivatized monosaccharides separated by hydrophilic interaction liquid chromatography.

If the mutarotation should be suppressed, chromatography on the reversed phase can be followed and usage of a solvent with a higher content of organic phase might be preferable. A full separation of such isomeric structures (anomers) was achieved hitherto by means of chiral HPLC (Lopes & Gaspar, 2008), ion-exchange chromatography or amino-columns (Schumacher & Kroh, 1995; Nishikawa *et al.*, 1996; Moriyasu *et al.*, 1984). More recently, separation of metal adducts of anomeric methyl glycoside isomers was achieved by ion mobility mass spectrometry (Dwivedi *et al.*, 2007).

Liquid Chromatography –

of BACH monosaccharides showed

ions.

Mass Spectrometry of Carbohydrates Derivatized with Biotinamidocaproyl Hydrazide 69

The protonated ion species of the BACH-derivatized monosaccharides showed under the MS-fragmentation multiple water losses from the molecular ion (100%), 0,2X-fragment at m/z 414 (~20%) and some abundant cleavages associated with the BACH label: m/z 372 (100%) – detachment of the whole label, m/z 357 (~23%) – loss of an ammonia group and m/z 227

similarity with the fragmentation spectrum of the [M+Na]+- ion and yielded weak 0,2X-ring cleavage (m/z 412, ~7%) and the abundant cleavage of the hydrazine label (m/z 355, 100%). In the case of *N*-acetylglucosamine, on the other hand, the 0,2X-fragment was the only prominent peak in the MS/MS spectrum with 100% of rel. intensity. Hence, variation in abundances of fragment-ions of the aminosugar GlcNAc in comparison with fragments of

(~43%) which presents the acylium ion of the biotin component (Fig. 10).

neutral sugars was observed when performing CID of [M+Na]+ and [M-H]-

**3.4. Hydrophilic interaction liquid chromatography of BACH-derivatized**

A common obstacle in the mass spectrometry of saccharides is the characterization of the stereochemistry of the glycosidic bond (Zhu *et al.*, 2009; Morelle & Michalski, 2005). Isomers usually have very similar MS2- or MS3-fragmentation and are difficult to differentiate without a previous separation. Therefore, the MS discrimination of isomers is often aided by

The formation of closed-ring derivatives (glycosylhydrazides) through non-reducing derivatization has demonstrated some advantages in the chromatographic separation and in the analysis of isomers (Li & Her, 1998, 1993). Also, differentiation of anomeric configuration has been observed in some cases following derivatization (Xue *et al.*, 2004; Ashline *et al.*, 2005). Clowers et al. achieved this with reduced form of isobaric di- and trisaccharides by ion mobility mass spectrometry (Clowers *et al.*, 2005). These research groups exploring isomeric sugars with the focus on their MS fragmentation, observed differences in the negative-ion electrospray mass spectra of structurally related compounds. The presence/absence and abundance of certain fragment ions allowed for an assembly of empirical criteria for assigning either a linkage position or the anomeric configuration of a disaccharide. For example, diagnostic ions (m/z 221, 263, 281) which were characteristic for 1-4-linkage were observed by negative ion mode mass spectrometry (Mulroney *et al.*, 1995; Garozzo *et al.*, 1991). With glucose-containing disaccharides, dissociation of the m/z 221 generated product ions that allowed for the differentiation of anomeric configuration (Fang

Here we sought to examine whether the BACH-derivatized isomeric disaccharides would be separable on HILIC and would in MS/MS dissociate to yield product ions from which stereochemical information might be obtained about the glycosidic bond. For this purpose, maltose (α-D-Glc-(1-4)-D-Glc) and cellobiose (β-D-Glc-(1-4)-D-Glc), sugars which vary solely in their anomeric configuration were derivatized by non-reducing BACH-labeling according

The deprotonated form of the molecular ion [M-H]-

one or more orthogonal pre-mass separation techniques.

& Bendiak, 2007; Fang *et al.*, 2007).

to the protocol described in the experimental part.

**disaccharides**

## *3.3.1. MS/MS of BACH-derivatized monosaccharides*

The BACH-derivatized monosaccharides were subjected to tandem mass spectrometry. Analysis by ESI-ion trap showed that sodium adducts were the most abundant ions. Less abundant [M+H]+ ions were detected in positive ion mode as well. Negative ion mode MS yielded [M-H] ions of derivatized monosaccharides. All three types of ions were subjected to MS/MS in order to examine their fragmentation (Fig. 10).

MS/MS of sodium adducts [M+Na]+ of neutral monosaccharides provided X-ring-cleavage in position 0-2 (m/z 436, 10% rel. intensity (RI)) and cleavage between two nitrogen atoms of the hydrazide group with m/z 379 (100% RI). With the sodiated ion of BACH-derivatized aminosugar *N*-acetylglucosamine, both of the cleavages were abundantly present in the spectrum (0,2X at m/z 477, 100% RI; m/z 379, ~60% RI) and were accompanied by minor water losses from the molecular ion.

**Figure 10.** Scheme of fragmentation observed in MS/MS of BACH-labeled monosaccharides with different types of ions. Nominal masses are given.

The protonated ion species of the BACH-derivatized monosaccharides showed under the MS-fragmentation multiple water losses from the molecular ion (100%), 0,2X-fragment at m/z 414 (~20%) and some abundant cleavages associated with the BACH label: m/z 372 (100%) – detachment of the whole label, m/z 357 (~23%) – loss of an ammonia group and m/z 227 (~43%) which presents the acylium ion of the biotin component (Fig. 10).

68 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

mobility mass spectrometry (Dwivedi *et al.*, 2007).

yielded [M-H]-

water losses from the molecular ion.

different types of ions. Nominal masses are given.

*3.3.1. MS/MS of BACH-derivatized monosaccharides*

to MS/MS in order to examine their fragmentation (Fig. 10).

If the mutarotation should be suppressed, chromatography on the reversed phase can be followed and usage of a solvent with a higher content of organic phase might be preferable. A full separation of such isomeric structures (anomers) was achieved hitherto by means of chiral HPLC (Lopes & Gaspar, 2008), ion-exchange chromatography or amino-columns (Schumacher & Kroh, 1995; Nishikawa *et al.*, 1996; Moriyasu *et al.*, 1984). More recently, separation of metal adducts of anomeric methyl glycoside isomers was achieved by ion

The BACH-derivatized monosaccharides were subjected to tandem mass spectrometry. Analysis by ESI-ion trap showed that sodium adducts were the most abundant ions. Less abundant [M+H]+ ions were detected in positive ion mode as well. Negative ion mode MS

MS/MS of sodium adducts [M+Na]+ of neutral monosaccharides provided X-ring-cleavage in position 0-2 (m/z 436, 10% rel. intensity (RI)) and cleavage between two nitrogen atoms of the hydrazide group with m/z 379 (100% RI). With the sodiated ion of BACH-derivatized aminosugar *N*-acetylglucosamine, both of the cleavages were abundantly present in the spectrum (0,2X at m/z 477, 100% RI; m/z 379, ~60% RI) and were accompanied by minor

**Figure 10.** Scheme of fragmentation observed in MS/MS of BACH-labeled monosaccharides with

ions of derivatized monosaccharides. All three types of ions were subjected

The deprotonated form of the molecular ion [M-H] of BACH monosaccharides showed similarity with the fragmentation spectrum of the [M+Na]+- ion and yielded weak 0,2X-ring cleavage (m/z 412, ~7%) and the abundant cleavage of the hydrazine label (m/z 355, 100%). In the case of *N*-acetylglucosamine, on the other hand, the 0,2X-fragment was the only prominent peak in the MS/MS spectrum with 100% of rel. intensity. Hence, variation in abundances of fragment-ions of the aminosugar GlcNAc in comparison with fragments of neutral sugars was observed when performing CID of [M+Na]+ and [M-H] ions.

## **3.4. Hydrophilic interaction liquid chromatography of BACH-derivatized disaccharides**

A common obstacle in the mass spectrometry of saccharides is the characterization of the stereochemistry of the glycosidic bond (Zhu *et al.*, 2009; Morelle & Michalski, 2005). Isomers usually have very similar MS2- or MS3-fragmentation and are difficult to differentiate without a previous separation. Therefore, the MS discrimination of isomers is often aided by one or more orthogonal pre-mass separation techniques.

The formation of closed-ring derivatives (glycosylhydrazides) through non-reducing derivatization has demonstrated some advantages in the chromatographic separation and in the analysis of isomers (Li & Her, 1998, 1993). Also, differentiation of anomeric configuration has been observed in some cases following derivatization (Xue *et al.*, 2004; Ashline *et al.*, 2005). Clowers et al. achieved this with reduced form of isobaric di- and trisaccharides by ion mobility mass spectrometry (Clowers *et al.*, 2005). These research groups exploring isomeric sugars with the focus on their MS fragmentation, observed differences in the negative-ion electrospray mass spectra of structurally related compounds. The presence/absence and abundance of certain fragment ions allowed for an assembly of empirical criteria for assigning either a linkage position or the anomeric configuration of a disaccharide. For example, diagnostic ions (m/z 221, 263, 281) which were characteristic for 1-4-linkage were observed by negative ion mode mass spectrometry (Mulroney *et al.*, 1995; Garozzo *et al.*, 1991). With glucose-containing disaccharides, dissociation of the m/z 221 generated product ions that allowed for the differentiation of anomeric configuration (Fang & Bendiak, 2007; Fang *et al.*, 2007).

Here we sought to examine whether the BACH-derivatized isomeric disaccharides would be separable on HILIC and would in MS/MS dissociate to yield product ions from which stereochemical information might be obtained about the glycosidic bond. For this purpose, maltose (α-D-Glc-(1-4)-D-Glc) and cellobiose (β-D-Glc-(1-4)-D-Glc), sugars which vary solely in their anomeric configuration were derivatized by non-reducing BACH-labeling according to the protocol described in the experimental part.

Mass Spectrometry of Carbohydrates Derivatized with Biotinamidocaproyl Hydrazide 71

Negative MS/MS fragmentation spectra of BACH-labeled maltose and cellobiose were similar in terms of presence of diagnostic ions. However, some variation in the relative abundance of the product ions was visible. Ions observed were: m/z 179, 221, 263, 281 (Fig. 12), where m/z 179 represents the non-reducing monosaccharide containing the glycosidic oxygen; m/z 221 (2,4A2) comprises intact non-reducing sugar glycosidically linked to a glycoaldehyde molecule and m/z 281 corresponds to loss of one –CHOH backbone-unit and also one -CH with the BACH-label. This ion eliminated a molecule of water yielding an m/z 263 ion. This has been shown to be characteristic for 1,4-bonded underivatized glucose

In the spectrum of α-linked maltose (Fig. 13a), intensity of the m/z 221 was slightly higher than m/z 263. In the fragmentation spectra of beta-linked cellobiose, on the contrary, the ion m/z 221 appeared only in traces. With cellobiose, the intensity of m/z 263 was always higher than m/z 221. Further, the ion at m/z 281 was more abundant in the spectrum of maltose, whereas almost absent in the MS/MS of cellobiose. In order to confirm the reproducibility of the fragmentation we performed the measurements 15 times in total (5 rounds of measurements for each disaccharide on three different days) indicating the presence and intensity of respective fragments to be reproducible and constant. These observations are in accordance with the fragments reported for 4-linked disaccharides and their proportionalities correspond with dissociation studies reported for the differentiation of this type of isomeric disaccharides. In the study of Li and Her (Li & Her, 1993), where relative abundance of fragment ions of 1-4 linked disaccharides was compared, the intensity of the m/z 221 was higher than m/z 263 in alpha-linked disaccharides and smaller than m/z 263 ion in beta-linked disaccharides. As reported (Fang & Bendiak, 2007; Fang *et al.*, 2007), the cross-ring cleavage m/z 221 of the disaccharides also showed to be useful in the discrimination of anomeric configuration. Under conditions applied here, further dissociation of m/z 221 (MS3) of the BACH- labeled maltose or cellobiose provided no additional differential information. Noneless, through the combination of BACH-labeling and tandem mass spectrometry, distinctive structural information was obtained at the MS2-stage of fragmentation and disaccharides with the same linkage but different anomeric configurations (maltose: 1-4-α-linkage, cellobiose: 1-4-β-linkage) could have

**Figure 12.** Fragmentation of [M-H]-

dimers (Dallinga & Heerma, 1991a).

been distinguished.

cellobiose in negative ion mode

**Figure 11.** TIC showing separation of BACH-labeled isomeric disaccharides by means of hydrophilic interaction liquid chromatography. Peak assignements: 1 = BACH-maltose, 2 = BACH-cellobiose, \* isomeric byproduct upon derivatization when BACH-label used in original unpurified form, BACH = excess of labeling reagent

With underivatized maltose and cellobiose, HPLC-separation and signal intensity were very poor (data not shown). Through BACH-derivatization, enhancement of signal intensity of two orders of magnitude (from 2x105 to 6x107) in comparison with native counterparts was achieved and at least a partial separation on the HILIC-phase was reached (Fig. 11). Even if they were not completely resolved, this demonstrated the feasibility of determining the presence of isomeric hits. For MS/MS investigation, the sugars were subjected separately to mass spectrometry, in order to obtain discrete fragmentation data originating only from one of the isomers. MS/MS spectra of underivatized maltose and cellobiose were without any significant difference in fragmentation pattern. Thus, no assignment could have been done with the native sugars.

In the study of BACH-labeled maltose and cellobiose, different types of molecular ions were detected: [M+Na]+ (m/z 718), [M+H]+ (m/z 696) and [M-H]- (m/z 694). These were subsequently subjected to tandem mass spectrometry in order to examine their fragmentation profile. Each of the precursor ions showed different fragmentation:

In positive ion mode, same fragmentation patterns for maltose and cellobiose were observed (data not shown). MS/MS of the sodium adduct contained the 0,2X-ring cleavage (m/z 436) from the reducing end of the derivatized disaccharide and the N-N-cleavage of the hydrazide group of the BACH-label (m/z 355). Fragmentation spectra of the [M+H]+-ion were similar to [M+Na]+. They provided week 0,2X-fragment (m/z 414) and the hydrazide group cleavage (m/z 355). Additionally to these, glycosidic cleavages (Y0 and Y1), which confirmed the sequence and hexose composition of the saccharide, appeared. With Y1-ion, up to four water losses were observed. Hence, in positive ion mode, when observing either [M+Na]+ or [M+H]+, no difference in the fragmentation of these isomers was observed.

CID of the [M-H]—ion, on the contrary (Fig. 12), yielded cleavages (m/z 412 and m/z 355) and also fragments between m/z 179 and m/z 355, having higher masses than monosaccharide and encompassing the glycosidic bond of the sugar.

Mass Spectrometry of Carbohydrates Derivatized with Biotinamidocaproyl Hydrazide 71

70 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

excess of labeling reagent

with the native sugars.

**Figure 11.** TIC showing separation of BACH-labeled isomeric disaccharides by means of hydrophilic interaction liquid chromatography. Peak assignements: 1 = BACH-maltose, 2 = BACH-cellobiose, \* isomeric byproduct upon derivatization when BACH-label used in original unpurified form, BACH =

With underivatized maltose and cellobiose, HPLC-separation and signal intensity were very poor (data not shown). Through BACH-derivatization, enhancement of signal intensity of two orders of magnitude (from 2x105 to 6x107) in comparison with native counterparts was achieved and at least a partial separation on the HILIC-phase was reached (Fig. 11). Even if they were not completely resolved, this demonstrated the feasibility of determining the presence of isomeric hits. For MS/MS investigation, the sugars were subjected separately to mass spectrometry, in order to obtain discrete fragmentation data originating only from one of the isomers. MS/MS spectra of underivatized maltose and cellobiose were without any significant difference in fragmentation pattern. Thus, no assignment could have been done

In the study of BACH-labeled maltose and cellobiose, different types of molecular ions were

subsequently subjected to tandem mass spectrometry in order to examine their

In positive ion mode, same fragmentation patterns for maltose and cellobiose were observed (data not shown). MS/MS of the sodium adduct contained the 0,2X-ring cleavage (m/z 436) from the reducing end of the derivatized disaccharide and the N-N-cleavage of the hydrazide group of the BACH-label (m/z 355). Fragmentation spectra of the [M+H]+-ion were similar to [M+Na]+. They provided week 0,2X-fragment (m/z 414) and the hydrazide group cleavage (m/z 355). Additionally to these, glycosidic cleavages (Y0 and Y1), which confirmed the sequence and hexose composition of the saccharide, appeared. With Y1-ion, up to four water losses were observed. Hence, in positive ion mode, when observing either [M+Na]+ or [M+H]+, no difference in the fragmentation of these isomers was observed.

CID of the [M-H]—ion, on the contrary (Fig. 12), yielded cleavages (m/z 412 and m/z 355) and also fragments between m/z 179 and m/z 355, having higher masses than

fragmentation profile. Each of the precursor ions showed different fragmentation:

(m/z 694). These were

detected: [M+Na]+ (m/z 718), [M+H]+ (m/z 696) and [M-H]-

monosaccharide and encompassing the glycosidic bond of the sugar.

**Figure 12.** Fragmentation of [M-H]- -ion observed for BACH-labeled isomeric disaccharides maltose and cellobiose in negative ion mode

Negative MS/MS fragmentation spectra of BACH-labeled maltose and cellobiose were similar in terms of presence of diagnostic ions. However, some variation in the relative abundance of the product ions was visible. Ions observed were: m/z 179, 221, 263, 281 (Fig. 12), where m/z 179 represents the non-reducing monosaccharide containing the glycosidic oxygen; m/z 221 (2,4A2) comprises intact non-reducing sugar glycosidically linked to a glycoaldehyde molecule and m/z 281 corresponds to loss of one –CHOH backbone-unit and also one -CH with the BACH-label. This ion eliminated a molecule of water yielding an m/z 263 ion. This has been shown to be characteristic for 1,4-bonded underivatized glucose dimers (Dallinga & Heerma, 1991a).

In the spectrum of α-linked maltose (Fig. 13a), intensity of the m/z 221 was slightly higher than m/z 263. In the fragmentation spectra of beta-linked cellobiose, on the contrary, the ion m/z 221 appeared only in traces. With cellobiose, the intensity of m/z 263 was always higher than m/z 221. Further, the ion at m/z 281 was more abundant in the spectrum of maltose, whereas almost absent in the MS/MS of cellobiose. In order to confirm the reproducibility of the fragmentation we performed the measurements 15 times in total (5 rounds of measurements for each disaccharide on three different days) indicating the presence and intensity of respective fragments to be reproducible and constant. These observations are in accordance with the fragments reported for 4-linked disaccharides and their proportionalities correspond with dissociation studies reported for the differentiation of this type of isomeric disaccharides. In the study of Li and Her (Li & Her, 1993), where relative abundance of fragment ions of 1-4 linked disaccharides was compared, the intensity of the m/z 221 was higher than m/z 263 in alpha-linked disaccharides and smaller than m/z 263 ion in beta-linked disaccharides. As reported (Fang & Bendiak, 2007; Fang *et al.*, 2007), the cross-ring cleavage m/z 221 of the disaccharides also showed to be useful in the discrimination of anomeric configuration. Under conditions applied here, further dissociation of m/z 221 (MS3) of the BACH- labeled maltose or cellobiose provided no additional differential information. Noneless, through the combination of BACH-labeling and tandem mass spectrometry, distinctive structural information was obtained at the MS2-stage of fragmentation and disaccharides with the same linkage but different anomeric configurations (maltose: 1-4-α-linkage, cellobiose: 1-4-β-linkage) could have been distinguished.

Mass Spectrometry of Carbohydrates Derivatized with Biotinamidocaproyl Hydrazide 73

MS2-stage of both trisaccharides and is due to the elimination of a site residue from the [M-

**Figure 14.** a) Separation of the mixture of two isomeric trisaccharides melezitose (1) and raffinose (2), demonstrating the value of coupling HILIC with ion-trap mass spectrometry performed to MS3. b) MS2

of melezitose c) MS2 of raffinose d) MS3 of melezitose e) MS3 of raffinose

H]-

Heerma, 1991; Perez-Victoria *et al.*, 2008).

**Figure 13.** MS/MS of BACH-derivatized isomeric disaccharides. a) Maltose-BACH b) Cellobiose-BACH

## **3.5. Hydrophilic interaction liquid chromatography of isomeric non-reducing trisaccharides**

In order to further examine the ability of the current analytical system to distinguish between the sugar isomers, we have analyzed two trisaccharides melezitose [α-D-Glc- (1→3)-β-D-Fru-(2→1)-α-D-Glc] and raffinose [α-D-Gal-(1→6)-α-D-Glc-(1→2)-β-D-Fru] which differ in sequence, linkage and anomeric configuration. Both of them were examined by LC-MS/MS in negative ion mode as this one showed to be the preferential mode in order to observe some distinctive fragmentation of related compounds (Section 3.4.). Figure 14a shows the total ion current of melezitose and raffinose. The chromatography of these sugars on the HILIC-phase provided a full baseline separation.

The fragmentation mass spectra of these trisaccharides were obtained from the deprotonated form of the molecular ion [M-H]- at m/z 503 (Figure 14b-e). Melezitose displayed a dominant fragment at m/z 323 which derives from cleavage of one of the two possible glycosidic bonds (Fig. 14b). This route was confirmed through the MS3 experiment, where the dissociation of m/z 341 generated no m/z 323 but solely the ions with m/z 179 and m/z 161 (Fig. 14d). This indicates that m/z 323 occurs directly through B-fragmentation and not via formation of m/z 341 and a subsequent water loss. The peak at m/z 341 was found at MS2-stage of both trisaccharides and is due to the elimination of a site residue from the [M-H]- -molecular ion. Interestingly, in positive-ion MS-studies on [M+H]+-ions of non-reducing oligosaccharides containing fructose (including melezitose and raffinose), the formation of m/z 323 was proposed to preferably occur via the water loss from m/z 341 (Dallinga & Heerma, 1991; Perez-Victoria *et al.*, 2008).

72 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

**Figure 13.** MS/MS of BACH-derivatized isomeric disaccharides. a) Maltose-BACH b) Cellobiose-BACH

In order to further examine the ability of the current analytical system to distinguish between the sugar isomers, we have analyzed two trisaccharides melezitose [α-D-Glc- (1→3)-β-D-Fru-(2→1)-α-D-Glc] and raffinose [α-D-Gal-(1→6)-α-D-Glc-(1→2)-β-D-Fru] which differ in sequence, linkage and anomeric configuration. Both of them were examined by LC-MS/MS in negative ion mode as this one showed to be the preferential mode in order to observe some distinctive fragmentation of related compounds (Section 3.4.). Figure 14a shows the total ion current of melezitose and raffinose. The chromatography of these sugars

The fragmentation mass spectra of these trisaccharides were obtained from the deprotonated form of the molecular ion [M-H]- at m/z 503 (Figure 14b-e). Melezitose displayed a dominant fragment at m/z 323 which derives from cleavage of one of the two possible glycosidic bonds (Fig. 14b). This route was confirmed through the MS3 experiment, where the dissociation of m/z 341 generated no m/z 323 but solely the ions with m/z 179 and m/z 161 (Fig. 14d). This indicates that m/z 323 occurs directly through B-fragmentation and not via formation of m/z 341 and a subsequent water loss. The peak at m/z 341 was found at

**3.5. Hydrophilic interaction liquid chromatography of isomeric non-reducing**

on the HILIC-phase provided a full baseline separation.

**trisaccharides**

**Figure 14.** a) Separation of the mixture of two isomeric trisaccharides melezitose (1) and raffinose (2), demonstrating the value of coupling HILIC with ion-trap mass spectrometry performed to MS3. b) MS2 of melezitose c) MS2 of raffinose d) MS3 of melezitose e) MS3 of raffinose

Raffinose fragmented to form preferably m/z 179, m/z 221 and glycosidic fragment at m/z 341 (Fig. 14c). Other ions (m/z 323 and 161) were present as well. At this stage of fragmentation (MS2), the formation of cross-ring cleavage (m/z 221) involving either the 0-2 or 0-4 bonds of the center residue appeared. This ion has not been seen in the spectrum of melezitose (compare to Fig. 14b). The same fact was reported in the studies of fragmentation of trisaccharides by negative FAB (Dallinga & Heerma, 1991b) and negative ion mobility mass spectrometry (Liu & Clemmer, 1997): raffinose yielded m/z 221 in the MS2-spectrum, whereas melezitose showed only the product ion at m/z 323 without presence of m/z 221. The presence of m/z 221 was also evident in MS3 of raffinose (Fig. 14e). The different fragmentation routes observed may be attributed to the difference in the steric character of these isomers: Melezitose has a branched character and raffinose represents a linear molecule.

Liquid Chromatography –

Mass Spectrometry of Carbohydrates Derivatized with Biotinamidocaproyl Hydrazide 75

BACH-derivatized isomeric disaccharides and to fully separate non-reducing isomeric

In terms of the enhancement of mass spectrometric signal of mono- and disaccharides, BACH-labeling provided higher intense ions than 2-AB-derivatization. Analysis of the BACH-derivatives in combination with the mass spectrometry can be pursued, when investigation of isomers is desired. Unlike positive ESI-MS, negative ion MS of BACHderivatized isomeric disaccharides provided ring-cleavage ions which were helpful in the differentiation of the anomeric configuration of the glycosidic bond. Further, separation of structurally related non-derivatized trisaccharides was achieved on HILIC and their differentiation on the basis of multiple stage mass spectrometry was possible. This differentiation would not have been possible without the suitable pre-mass spectrometric separation. In this way, coupling of the HILIC-separation with MSn-fragmentation might be a valuable approach on the way to the distinction of certain types of isomeric

*University of Würzburg/Department of Pharmacy and Food Chemistry, Germany*

This work was supported by the Fond der Chemischen Industrie and the Universitätsbund

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Ashline, D., Singh, S., Hanneman, A. & Reinhold, V. (2005). Congruent strategies for carbohydrate sequencing. 1. Mining structural details by MSn. *Analytical Chemistry,*

Bigge, J. C., Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M. & Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. *Analytical Biochemistry,* Vol.230, No.2, pp. 229-238, ISSN 0003-2697 Bindila, L. & Peter-Katalinic, J. (2009). Chip-mass spectrometry for glycomic studies. *Mass*

Brons, C. & Olieman, C. (1983). Study of the high-performance liquid-chromatographic separation of reducing sugars, applied to the determination of lactose in milk. *Journal of*

trisaccharides.

structures.

**Author details**

**Acknowledgement**

of University of Würzburg.

**5. References**

Petra Kapková and Stephanie Bank

No.4, pp. 495-505, ISSN 0951-14198

Vol.77, No.19, pp. 6250-6262, ISSN 0003-2700

*Spectrometry Reviews,* Vol.28, No.2, pp. 223-253, ISSN 1098-2787

*Chromatography,* Vol.259, No.1, pp. 79-86, ISSN 0021-9673

Finally, the differences in the chromatography on the HILIC-phase and in the fragmentation pattern (presence and rel. intensity of the product ions in the MS2 and MS3 stage) of the melezitose and raffinose indicated the occurrence of distinct isomeric structures.

This distinguished fragmentation would not have been possible to obtain without the previous pre-mass spectrometric separation on the HILIC-phase. Considering mass spectrometric techniques, such differential identification of these isomers out of their mixture was achieved up to now, only by means of ion-mobility mass spectrometry (Zhu *et al.*, 2009; Liu & Clemmer, 1997). To our best knowledge, this is the first report on separation and identification of such isomers by coupling of hydrophilic interaction chromatography and mass spectrometry.

## **4. Conclusion**

Different types of *N*-glycans (high-mannose, complex and hybrid) have been derivatized with biotinamidocaproyl hydrazide and analyzed by chip-based LC/ESI triple-quadrupole mass spectrometry. This approach allowed for rapid mass spectrometric selection of glycans out of glycan mixtures on the base of the scanning for a common feature ion (oxonium ion). The glycans observed upon MS were primarily in the doubly charged state. For each type of glycan, product ion spectra with the characteristic B- and Y-ions were observed which allowed for elucidation of the structure and segregation of the glycans into the respective class. This methodology provided fast structural information about the nature of the glycan pool present in a glycoprotein and may be used in the profiling of *N*-linked glycans. Beyond structural mass spectrometric studies, BACHderivatized glycans can potentially be used also in functional studies with carbohydrate binding proteins, as recently reported.

HILIC and reversed phase liquid chromatography of BACH- and 2-AB-labeled monosaccharides enabled the investigation of different analytical objectives and showed some orthogonal features in terms of the separation of monosaccharides frequently present in glycoproteins. The HILIC-system was not sufficient for separation of anomers of BACH-derivatized monosaccharides. It was, however, capable to partially resolve the BACH-derivatized isomeric disaccharides and to fully separate non-reducing isomeric trisaccharides.

In terms of the enhancement of mass spectrometric signal of mono- and disaccharides, BACH-labeling provided higher intense ions than 2-AB-derivatization. Analysis of the BACH-derivatives in combination with the mass spectrometry can be pursued, when investigation of isomers is desired. Unlike positive ESI-MS, negative ion MS of BACHderivatized isomeric disaccharides provided ring-cleavage ions which were helpful in the differentiation of the anomeric configuration of the glycosidic bond. Further, separation of structurally related non-derivatized trisaccharides was achieved on HILIC and their differentiation on the basis of multiple stage mass spectrometry was possible. This differentiation would not have been possible without the suitable pre-mass spectrometric separation. In this way, coupling of the HILIC-separation with MSn-fragmentation might be a valuable approach on the way to the distinction of certain types of isomeric structures.

## **Author details**

74 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

has a branched character and raffinose represents a linear molecule.

and mass spectrometry.

binding proteins, as recently reported.

**4. Conclusion**

Raffinose fragmented to form preferably m/z 179, m/z 221 and glycosidic fragment at m/z 341 (Fig. 14c). Other ions (m/z 323 and 161) were present as well. At this stage of fragmentation (MS2), the formation of cross-ring cleavage (m/z 221) involving either the 0-2 or 0-4 bonds of the center residue appeared. This ion has not been seen in the spectrum of melezitose (compare to Fig. 14b). The same fact was reported in the studies of fragmentation of trisaccharides by negative FAB (Dallinga & Heerma, 1991b) and negative ion mobility mass spectrometry (Liu & Clemmer, 1997): raffinose yielded m/z 221 in the MS2-spectrum, whereas melezitose showed only the product ion at m/z 323 without presence of m/z 221. The presence of m/z 221 was also evident in MS3 of raffinose (Fig. 14e). The different fragmentation routes observed may be attributed to the difference in the steric character of these isomers: Melezitose

Finally, the differences in the chromatography on the HILIC-phase and in the fragmentation pattern (presence and rel. intensity of the product ions in the MS2 and MS3 stage) of the

This distinguished fragmentation would not have been possible to obtain without the previous pre-mass spectrometric separation on the HILIC-phase. Considering mass spectrometric techniques, such differential identification of these isomers out of their mixture was achieved up to now, only by means of ion-mobility mass spectrometry (Zhu *et al.*, 2009; Liu & Clemmer, 1997). To our best knowledge, this is the first report on separation and identification of such isomers by coupling of hydrophilic interaction chromatography

Different types of *N*-glycans (high-mannose, complex and hybrid) have been derivatized with biotinamidocaproyl hydrazide and analyzed by chip-based LC/ESI triple-quadrupole mass spectrometry. This approach allowed for rapid mass spectrometric selection of glycans out of glycan mixtures on the base of the scanning for a common feature ion (oxonium ion). The glycans observed upon MS were primarily in the doubly charged state. For each type of glycan, product ion spectra with the characteristic B- and Y-ions were observed which allowed for elucidation of the structure and segregation of the glycans into the respective class. This methodology provided fast structural information about the nature of the glycan pool present in a glycoprotein and may be used in the profiling of *N*-linked glycans. Beyond structural mass spectrometric studies, BACHderivatized glycans can potentially be used also in functional studies with carbohydrate

HILIC and reversed phase liquid chromatography of BACH- and 2-AB-labeled monosaccharides enabled the investigation of different analytical objectives and showed some orthogonal features in terms of the separation of monosaccharides frequently present in glycoproteins. The HILIC-system was not sufficient for separation of anomers of BACH-derivatized monosaccharides. It was, however, capable to partially resolve the

melezitose and raffinose indicated the occurrence of distinct isomeric structures.

Petra Kapková and Stephanie Bank *University of Würzburg/Department of Pharmacy and Food Chemistry, Germany*

## **Acknowledgement**

This work was supported by the Fond der Chemischen Industrie and the Universitätsbund of University of Würzburg.

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**Chapter 5**

© 2012 Jampílek and Dohnal, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Jampílek and Dohnal, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Investigation of Carbohydrates and Their**

Development in the field of pharmaceutical administration has resulted in the discovery of highly sophisticated drug delivery systems that allow for the maintenance of a constant drug level in an organism. Contrary to these revolution biopharmaceutical results, over the last ten years, the number of poorly soluble drugs has steadily increased. Estimates state that 40% of the drugs in the pipelines have solubility problems. Progress in high throughput screening methods leads to an even greater amount of newly discovered drugs, but a lot of them have poor water solubility. Literature states that about 60% of all drugs coming directly from synthesis are nowadays poorly soluble. Meanwhile the five key physicochemical properties, such as pKa, solubility, permeability, stability and lipophilicity, in early compound screening should be optimized. Compounds with insufficient solubility carry a higher risk of failure during discovery and development, since insufficient solubility may compromise other property assays, mask additional undesirable properties, influence both pharmacokinetic and pharmacodynamic properties of the compound and finally may affect the developability of the compound. Poor solubility in water correlates with poor bioavailability. If there is no way to improve drug solubility, it will not be able to be absorbed from the gastrointestinal tract into the bloodstream and reach the site of action

Modification/optimization of unfavourable physico-chemical properties of these drugs is possible through increasing their water solubility or improving permeability. There are many ways to solubilize certain poorly soluble drugs. But these methods are limited to drugs with certain properties in regard to their chemistry or, for example, to their molecular

Aqueous solubility can be increased by chemical exchange: (*i*) salts, co-crystals or solvates formation (affects also chemical stability, polymorphism, technological workability);

**Derivatives as Crystallization Modifiers**

Josef Jampílek and Jiří Dohnal

http://dx.doi.org/10.5772/50626

**1. Introduction**

size or conformation.

Additional information is available at the end of the chapter

(Junghanns & Müller, 2008; Payghan et al., 2008).

Zhu, M. L., Bendiak, B., Clowers, B. & Hill, H. H. (2009). Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions. *Analytical and Bioanalytical Chemistry,* Vol.394, No.7, pp. 1853-1867, ISSN 1618-2650

## **Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers**

Josef Jampílek and Jiří Dohnal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50626

## **1. Introduction**

80 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

Vol.394, No.7, pp. 1853-1867, ISSN 1618-2650

*Spectrometry,* Vol.18, No.17, pp. 1947-1955, ISSN 0951-4198

using electrospray ionization tandem mass spectrometry. *Rapid Communications in Mass*

Zhu, M. L., Bendiak, B., Clowers, B. & Hill, H. H. (2009). Ion mobility-mass spectrometry analysis of isomeric carbohydrate precursor ions. *Analytical and Bioanalytical Chemistry,*

> Development in the field of pharmaceutical administration has resulted in the discovery of highly sophisticated drug delivery systems that allow for the maintenance of a constant drug level in an organism. Contrary to these revolution biopharmaceutical results, over the last ten years, the number of poorly soluble drugs has steadily increased. Estimates state that 40% of the drugs in the pipelines have solubility problems. Progress in high throughput screening methods leads to an even greater amount of newly discovered drugs, but a lot of them have poor water solubility. Literature states that about 60% of all drugs coming directly from synthesis are nowadays poorly soluble. Meanwhile the five key physicochemical properties, such as pKa, solubility, permeability, stability and lipophilicity, in early compound screening should be optimized. Compounds with insufficient solubility carry a higher risk of failure during discovery and development, since insufficient solubility may compromise other property assays, mask additional undesirable properties, influence both pharmacokinetic and pharmacodynamic properties of the compound and finally may affect the developability of the compound. Poor solubility in water correlates with poor bioavailability. If there is no way to improve drug solubility, it will not be able to be absorbed from the gastrointestinal tract into the bloodstream and reach the site of action (Junghanns & Müller, 2008; Payghan et al., 2008).

> Modification/optimization of unfavourable physico-chemical properties of these drugs is possible through increasing their water solubility or improving permeability. There are many ways to solubilize certain poorly soluble drugs. But these methods are limited to drugs with certain properties in regard to their chemistry or, for example, to their molecular size or conformation.

> Aqueous solubility can be increased by chemical exchange: (*i*) salts, co-crystals or solvates formation (affects also chemical stability, polymorphism, technological workability);

© 2012 Jampílek and Dohnal, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Jampílek and Dohnal, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(*ii*) substitution by hydrophilic groups (effect of drugs with small molecules can be decreased); (*iii*) prodrug preparation (hydrolyzable amides or semiesters with polybasic acids). In general, the following structural modifications are the best way to improve permeability: (*i*) replacement of ionisable groups by non-ionizable groups; (*ii*) increase of lipophilicity; (*iii*) isosteric replacement of polar groups; (*iv*) esterification of carboxylic acid; (*v*) reduction of hydrogen bonding and polarity; (*vi*) reduction of size; (*vii*) addition of a non-polar side chain; (*viii*) preparation of prodrugs. Generally, these strategies are based on a few fundamental concepts: change of ionizability, lipophilicity, polarity or change of hydrogen bond donors or acceptors. Both approaches interact logically.

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 83

It should be made clear that no one particular strategy offers a solution for property enhancement of all APIs. Each API must be examined and evaluated on a case-by-case basis in terms of molecular structure and desired final properties (Schultheiss & Newman, 2009).

The term "polymorphism" (from Greek: *polys* = many, *morfé* = form) was first used by Mitscherlich in 1822. He noticed that one compound of a certain chemical composition can crystallize into several crystal forms (Mitscherlich, 1822). Polymorphism is an ability of substances to exist in two or more crystal modifications differing from each other by structure and/or molecule conformation in the crystal lattice. The concept of polymorphism is often confounded with isomorphism or pseudopolymorphism. These concepts are

In contrast to polymorphism when one substance able to form different crystal modifications is considered, in case of isomorphism two or more different substances that have just similar structure but form the same crystal modifications are considered. Such substances can even form so-called isomorphous series. Most often they originate as a result of co-crystallization of isomorphous substances from the mixture of their saturated solutions. A typical example is sulphates: magnesium sulphate, zinc sulphate and nickel sulphate crystallizing as heptahydrates. Also pseudopolymorphism should be distinguished from polymorphism. The concept of pseudopolymorphism is used for crystalline forms which also comprise solvent molecules as an integral part of their structure. These pseudopolymorphs are sometimes

The fundamental forms of solid substances can be classified into polymorphs, solvates/desolvated solvates and amorphous substances. A co-crystal can be defined as a multiple-component crystal, in which two or more molecules that are solid under ambient conditions coexist through a hydrogen bond (see Fig. 1 and Fig. 2). Polymorphism can be

**SOLIDS**

solvates

co-crystals

salts

single-component crystals multiple-component crystals

**Figure 1.** API solid form classification based on structure and composition. (Ref. Sekhon, 2009) (Taken

and adapted with the permission of the author.)

called solvates or hydrates if the solvent is water (Rodríguez-Spong et al., 2004).

amorphous structures crystals – polymorphs

interconnected but there are great differences between them.

**2. Polymorphism**

Based on these facts, pre-formulation/formulation can be another and mostly successful strategy for improving aqueous solubility and/or permeability and subsequently bioavailability. For example, selection of a suitable salt, particle size reduction (till nano size) connected with an increase of the surface area, change of polymorphic forms, selection of appropriate excipients to function as solubilizers/transporters (surfactants or pharmaceutical complexing agents, permeability enhancers) can be used for the oral dosage form (Kerns & Di, 2008).

It is well-known that crystalline materials obtain their fundamental physical properties from the molecular arrangement within the solid, and altering the placement and/or interactions between these molecules can, and usually does, have a direct impact on the properties of the particular solid. Currently, solid-state chemists call upon a variety of different strategies when attempting to alter the chemical and physical solid-state properties of active pharmaceutical ingredients (APIs), namely, the formation of salts, polymorphs, hydrates, solvates and co-crystals (Seddon & Zaworotko, 1999; Datta & Grant, 2004; Grepioni & Braga, 2007; Schultheiss & Newman, 2009).

Currently, salt formation is one of the primary solid-state approaches used to modify the physical properties of APIs, and it is estimated that over half of the medicines on the market are administered as salts (Bighley et al., 1996; Stahl & Wermuth, 2002; Gu & Grant, 2003.). However, a major limitation within this approach is that the API must possess a suitable (basic or acidic) ionizable site. In comparison, co-crystals (multi-component assemblies held together by freely reversible, non-covalent interactions) offer a different pathway, where any API regardless of acidic, basic or ionizable groups, could potentially be co-crystallized. This aspect helps to complement existing methods by reintroducing molecules that had limited pharmaceutical profiles based on their non-ionizable functional groups. Since the primary structure of a drug molecule does not change in co-crystals, their development in terms of the regulation "New Chemical Entities (NCEs)" of the U.S. Food and Drug Administration (FDA) as well as European Commission Regulation (EC) No. 258/97 carries fewer risks and takes much less time; nevertheless stability and bioequivalent studies are still necessary. In addition, the number of potential non-toxic co-crystal formers (or co-formers) that can be incorporated into a co-crystalline reaction is numerous, *e.i.* pharmaceutical excipients, amino acids, food additives, nutraceuticals, see for example the GRAS list (Generally Regarded as Safe) or the EAFUS Database (Everything Added to Food in the United States), both published by the FDA.

It should be made clear that no one particular strategy offers a solution for property enhancement of all APIs. Each API must be examined and evaluated on a case-by-case basis in terms of molecular structure and desired final properties (Schultheiss & Newman, 2009).

## **2. Polymorphism**

82 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

hydrogen bond donors or acceptors. Both approaches interact logically.

Di, 2008).

2007; Schultheiss & Newman, 2009).

published by the FDA.

(*ii*) substitution by hydrophilic groups (effect of drugs with small molecules can be decreased); (*iii*) prodrug preparation (hydrolyzable amides or semiesters with polybasic acids). In general, the following structural modifications are the best way to improve permeability: (*i*) replacement of ionisable groups by non-ionizable groups; (*ii*) increase of lipophilicity; (*iii*) isosteric replacement of polar groups; (*iv*) esterification of carboxylic acid; (*v*) reduction of hydrogen bonding and polarity; (*vi*) reduction of size; (*vii*) addition of a non-polar side chain; (*viii*) preparation of prodrugs. Generally, these strategies are based on a few fundamental concepts: change of ionizability, lipophilicity, polarity or change of

Based on these facts, pre-formulation/formulation can be another and mostly successful strategy for improving aqueous solubility and/or permeability and subsequently bioavailability. For example, selection of a suitable salt, particle size reduction (till nano size) connected with an increase of the surface area, change of polymorphic forms, selection of appropriate excipients to function as solubilizers/transporters (surfactants or pharmaceutical complexing agents, permeability enhancers) can be used for the oral dosage form (Kerns &

It is well-known that crystalline materials obtain their fundamental physical properties from the molecular arrangement within the solid, and altering the placement and/or interactions between these molecules can, and usually does, have a direct impact on the properties of the particular solid. Currently, solid-state chemists call upon a variety of different strategies when attempting to alter the chemical and physical solid-state properties of active pharmaceutical ingredients (APIs), namely, the formation of salts, polymorphs, hydrates, solvates and co-crystals (Seddon & Zaworotko, 1999; Datta & Grant, 2004; Grepioni & Braga,

Currently, salt formation is one of the primary solid-state approaches used to modify the physical properties of APIs, and it is estimated that over half of the medicines on the market are administered as salts (Bighley et al., 1996; Stahl & Wermuth, 2002; Gu & Grant, 2003.). However, a major limitation within this approach is that the API must possess a suitable (basic or acidic) ionizable site. In comparison, co-crystals (multi-component assemblies held together by freely reversible, non-covalent interactions) offer a different pathway, where any API regardless of acidic, basic or ionizable groups, could potentially be co-crystallized. This aspect helps to complement existing methods by reintroducing molecules that had limited pharmaceutical profiles based on their non-ionizable functional groups. Since the primary structure of a drug molecule does not change in co-crystals, their development in terms of the regulation "New Chemical Entities (NCEs)" of the U.S. Food and Drug Administration (FDA) as well as European Commission Regulation (EC) No. 258/97 carries fewer risks and takes much less time; nevertheless stability and bioequivalent studies are still necessary. In addition, the number of potential non-toxic co-crystal formers (or co-formers) that can be incorporated into a co-crystalline reaction is numerous, *e.i.* pharmaceutical excipients, amino acids, food additives, nutraceuticals, see for example the GRAS list (Generally Regarded as Safe) or the EAFUS Database (Everything Added to Food in the United States), both The term "polymorphism" (from Greek: *polys* = many, *morfé* = form) was first used by Mitscherlich in 1822. He noticed that one compound of a certain chemical composition can crystallize into several crystal forms (Mitscherlich, 1822). Polymorphism is an ability of substances to exist in two or more crystal modifications differing from each other by structure and/or molecule conformation in the crystal lattice. The concept of polymorphism is often confounded with isomorphism or pseudopolymorphism. These concepts are interconnected but there are great differences between them.

In contrast to polymorphism when one substance able to form different crystal modifications is considered, in case of isomorphism two or more different substances that have just similar structure but form the same crystal modifications are considered. Such substances can even form so-called isomorphous series. Most often they originate as a result of co-crystallization of isomorphous substances from the mixture of their saturated solutions. A typical example is sulphates: magnesium sulphate, zinc sulphate and nickel sulphate crystallizing as heptahydrates. Also pseudopolymorphism should be distinguished from polymorphism. The concept of pseudopolymorphism is used for crystalline forms which also comprise solvent molecules as an integral part of their structure. These pseudopolymorphs are sometimes called solvates or hydrates if the solvent is water (Rodríguez-Spong et al., 2004).

The fundamental forms of solid substances can be classified into polymorphs, solvates/desolvated solvates and amorphous substances. A co-crystal can be defined as a multiple-component crystal, in which two or more molecules that are solid under ambient conditions coexist through a hydrogen bond (see Fig. 1 and Fig. 2). Polymorphism can be

**Figure 1.** API solid form classification based on structure and composition. (Ref. Sekhon, 2009) (Taken and adapted with the permission of the author.)

classified depending on the fact if the molecule occurs in different conformations or is the same. If the molecule can have different conformations that crystallize differently and so the molecule is flexible, conformation polymorphism is observed. If the molecule is rigid, does not have any conformations and polymorphs differ only by their packing in the crystalline structure, this is the case of package polymorphism (Sharma et al., 2011).

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 85

During crystallization a self-assembly supramolecular process takes place when randomly oriented molecules assemble into internally structured crystals (Kratochvíl, 2007). There are several ways of crystallization: (*i*) by evaporation of solvent; (*ii*) by addition of antisolvent (product precipitation); (*iii*) by cooling of solution; (*iv*) by change of pH ; (*v*) by addition of a compound that will produce the desired product by a chemical reaction; (*vi*) by lyophilisation, sublimation or cooling of melt (Kratochvíl, 2010). The first two possibilities

The process of crystal formation is a complicated kinetic process which, if we simplify, is composed of nucleation, subsequent crystal growth around the formed crystal nuclei and growth termination. This happens at precipitation of a solid substance in the crystalline

The basis of nucleation is the quickest formation of the crystal nucleus. By constant sequence of molecules addition molecular aggregates, clusters, originate. The aggregates spontaneously impact and thus disintegrate but also grow. When they achieve the critical size, they become nuclei that do not disintegrate spontaneously but, to the contrary, only grow into a crystal. Nuclei can be classified into primary and secondary. Primary nucleation is further divided into homogenous and heterogeneous. Homogenous nucleation is characterized by formation of nuclei as a result of random impacts of aggregates somewhere in the solution volume without the presence of any foreign matter. By contrast, heterogeneous nucleation is assisted by foreign matters, for example, a stirrer. Secondary nucleation is also called seeded nucleation – small crystals (nuclei, seeds) of a desired crystal are added to the original solution. Seeding is successfully applied for systems where polymorphous behaviour cannot be excluded. Seeding of the system with a desired polymorph assures that an undesired polymorph will not be formed. After formation of the nucleus the system pass to the second mentioned crystallization phase. In this phase growth of crystals from the formed nucleus continues. If an API is crystallized with a co-crystallization partner, for example excipient or another API, with co-

For polymorphic drugs, the most thermodynamically stable polymorph is usually preferred. This polymorph normally assures reproducible bioavailability for the whole period of the pharmaceutical shelf life under different storing conditions that are possible in practice. A significant advantage is that production of such a polymorph is mostly easier controlled on an industrial scale. But in some cases a metastable crystalline form or an amorphous form is preferred due to patent and medical reasons. This happens when a higher concentration of an API in the system or faster dissolution of poorly soluble substances is required. Certain substances are used in the amorphous form also due to the fact that a crystalline form of the compound was not obtained. When an amorphous form or a metastable crystalline polymorph is used, especial attention is to be paid to safety assurance and the effectiveness of the drug for the whole shelf life period. These aspects are to be assured also for storing

**2.1. Crystallization**

were used in the experimental part.

form from solution or at solidification of a substance.

crystals formation, such process is called co-crystallization.

**2.2. Properties of solid forms**

conditions in other climatic zones.

Polymorphism should be distinguished from crystal morphology that represents crystallization of a substance from different solvents with a change of the form but without modification of the crystalline structure.

**Figure 2.** Schematic illustration of polymorphism of solid compounds. (Ref. Kratochvíl, 2009) (Taken and adapted from the presentation "Pharmaceutical Co-crystals" with the permission of the author.)

In the pharmaceutical practice it is conventional to collectively call all polymorphs, solvates and polyamorphs of the same API as solid forms or polymorphs in the extended meaning and designate them by, for example, Roman numerals or letters. It should be taken into consideration that designation of polymorphs is often non-uniform; it originates historically; therefore it is possible that two different polymorphs will be designated by different authors in the same way. A problem of pharmaceutical producers is polymorphic transitions in organic systems that are often hysteresis, badly defined and solventmediated. Polymorphic transitions can occur at all technological stages of drug production (at final API crystallization, wet granulation, micronization, tabletting or in ready tablets, for example, under the influence of auxiliaries – excipients). At drug registration, national regulation authorities demand description of all solid forms and possible phase transitions of the drug as well as a prescribed guarantee of the product polymorphic purity from pharmaceutical companies. At present approximately 85% of pharmaceutical products are solid formulations; therefore no manufacturer can afford to ignore the issue of polymorphism.

## **2.1. Crystallization**

84 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

modification of the crystalline structure.

polymorphism.

structure, this is the case of package polymorphism (Sharma et al., 2011).

classified depending on the fact if the molecule occurs in different conformations or is the same. If the molecule can have different conformations that crystallize differently and so the molecule is flexible, conformation polymorphism is observed. If the molecule is rigid, does not have any conformations and polymorphs differ only by their packing in the crystalline

Polymorphism should be distinguished from crystal morphology that represents crystallization of a substance from different solvents with a change of the form but without

**Figure 2.** Schematic illustration of polymorphism of solid compounds. (Ref. Kratochvíl, 2009) (Taken and adapted from the presentation "Pharmaceutical Co-crystals" with the permission of the author.)

In the pharmaceutical practice it is conventional to collectively call all polymorphs, solvates and polyamorphs of the same API as solid forms or polymorphs in the extended meaning and designate them by, for example, Roman numerals or letters. It should be taken into consideration that designation of polymorphs is often non-uniform; it originates historically; therefore it is possible that two different polymorphs will be designated by different authors in the same way. A problem of pharmaceutical producers is polymorphic transitions in organic systems that are often hysteresis, badly defined and solventmediated. Polymorphic transitions can occur at all technological stages of drug production (at final API crystallization, wet granulation, micronization, tabletting or in ready tablets, for example, under the influence of auxiliaries – excipients). At drug registration, national regulation authorities demand description of all solid forms and possible phase transitions of the drug as well as a prescribed guarantee of the product polymorphic purity from pharmaceutical companies. At present approximately 85% of pharmaceutical products are solid formulations; therefore no manufacturer can afford to ignore the issue of During crystallization a self-assembly supramolecular process takes place when randomly oriented molecules assemble into internally structured crystals (Kratochvíl, 2007). There are several ways of crystallization: (*i*) by evaporation of solvent; (*ii*) by addition of antisolvent (product precipitation); (*iii*) by cooling of solution; (*iv*) by change of pH ; (*v*) by addition of a compound that will produce the desired product by a chemical reaction; (*vi*) by lyophilisation, sublimation or cooling of melt (Kratochvíl, 2010). The first two possibilities were used in the experimental part.

The process of crystal formation is a complicated kinetic process which, if we simplify, is composed of nucleation, subsequent crystal growth around the formed crystal nuclei and growth termination. This happens at precipitation of a solid substance in the crystalline form from solution or at solidification of a substance.

The basis of nucleation is the quickest formation of the crystal nucleus. By constant sequence of molecules addition molecular aggregates, clusters, originate. The aggregates spontaneously impact and thus disintegrate but also grow. When they achieve the critical size, they become nuclei that do not disintegrate spontaneously but, to the contrary, only grow into a crystal. Nuclei can be classified into primary and secondary. Primary nucleation is further divided into homogenous and heterogeneous. Homogenous nucleation is characterized by formation of nuclei as a result of random impacts of aggregates somewhere in the solution volume without the presence of any foreign matter. By contrast, heterogeneous nucleation is assisted by foreign matters, for example, a stirrer. Secondary nucleation is also called seeded nucleation – small crystals (nuclei, seeds) of a desired crystal are added to the original solution. Seeding is successfully applied for systems where polymorphous behaviour cannot be excluded. Seeding of the system with a desired polymorph assures that an undesired polymorph will not be formed. After formation of the nucleus the system pass to the second mentioned crystallization phase. In this phase growth of crystals from the formed nucleus continues. If an API is crystallized with a co-crystallization partner, for example excipient or another API, with cocrystals formation, such process is called co-crystallization.

## **2.2. Properties of solid forms**

For polymorphic drugs, the most thermodynamically stable polymorph is usually preferred. This polymorph normally assures reproducible bioavailability for the whole period of the pharmaceutical shelf life under different storing conditions that are possible in practice. A significant advantage is that production of such a polymorph is mostly easier controlled on an industrial scale. But in some cases a metastable crystalline form or an amorphous form is preferred due to patent and medical reasons. This happens when a higher concentration of an API in the system or faster dissolution of poorly soluble substances is required. Certain substances are used in the amorphous form also due to the fact that a crystalline form of the compound was not obtained. When an amorphous form or a metastable crystalline polymorph is used, especial attention is to be paid to safety assurance and the effectiveness of the drug for the whole shelf life period. These aspects are to be assured also for storing conditions in other climatic zones.

Though the biological effect of an API is induced by interaction of the drug molecule with a target receptor when cell natural chemistry is influenced primarily by conformation changes, it is important in what solid phase the drug is administered to the patient. That means that not only the molecular but also the crystalline structure of an API is essential. The crystalline structure influences not only chemical and physical stability but also the drug dissolution rate and so can affect markedly the drug bioavailability. On the other hand, an amorphous form may have a considerably higher dissolution rate than the most stable polymorph. Great differences in the solubility and the dissolution rate of polymorphs can be a reason of significant differences in their distribution in the organism. Low plasma concentration can theoretically cause incomplete occupation of respective membrane receptors at the site of action (they are blocked by a substrate), or biological activity can change from agonist to antagonist or vice versa.

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 87

molecules in the same crystal structure (Stahly, 2007). Bhogala and Nangia define co-crystals as multi-component solid-state assemblies of two or more compounds held together by any type or combination of intermolecular interactions (Bhogala & Nangia, 2008). Childs and Hardcastle define co-crystals as a crystalline material made up of two or more components, usually in a stoichiometric ratio, each component being an atom, ionic compound, or molecule (Childs & Hardcastle, 2007). The simplest definition of co-crystals was proposed by Bond: "synonym for multi-component molecular crystal" (Bond, 2007). Also the definition of Aakeröy and Salmon can be recognized. They describe co-crystal as stoichiometric structurally homogeneous multi-component crystalline material of host-guest type that contains two or more neutral building blocks (discrete neutral molecular species that are solids at ambient conditions) that are present in definite amounts whereas all solids containing ions, including complex transition-metal ions, are excluded) (Aakeröy & Salmon, 2005). Jones defines co-crystal as a crystalline complex of two or more neutral molecular constituents bound together in the crystal structure through non-covalent interactions, often including hydrogen bonding (Jones et al., 2006) and Zaworotko says that co-crystals are formed between a molecular or ionic API and a co-crystal former that is a solid under

The host is an API, and the guest is a co-crystallization partner (an excipient or another API). Note that according to the definition by Aakeröy and Salmon co-crystals are different from solvates by the fact that the host and the guest of co-crystals are in the solid phase, while solvates contain both a solid phase and a solvent (or its residual), *i.e.* a liquid phase. However, there is still the problem of a boundary between the salt and the co-crystal, because according to the last mentioned definition of Aakeröy and Salmon, ions as co-crystal components are excluded, so on Fig. 3 only the first example of two is a co-crystal according to the mentioned definition. The borderline between salts and co-crystals is blurred and can be distinguished by the location of the proton between an acid and a base.

Several types of co-crystals can be distinguished: (*i*) "simple" co-crystals; (*ii*) solvated (hydrated) co-crystals – the co-crystal contains a component that is liquid at ambient temperature; (*iii*) salt co-crystals – the host is an ionized form; (*iv*) solvated salt co-crystals;

Pharmaceutical co-crystals, that is, co-crystals that are formed between an API and pharmaceutically acceptable (GRAS) compounds (co-crystal formers, counterions) that are solid under ambient conditions, represent a new paradigm in API formulation that might address important intellectual and physical property issues in the context of drug

Co-crystals can be understood as "addition compounds" or "organic molecular compounds". They are attractive to the pharmaceutical industry, because they offer opportunities to modify the chemical and/or physical properties of an API without the need to make or break covalent

This state can be denoted as salt–co-crystal continuum (Childs et al., 2007).

ambient conditions (Vishweshwar et al., 2006a).

(*v*) polymorphs of all previous types of co-crystals.

**3.1. Properties of co-crystals**

development and delivery.

However, the dissolution rate is not the only important parameter of the difference between polymorphs. There can be also differences in crystal size and shape that influence milling, tabletting, filtration, looseness and other important technological parameters. There are also differences in chemical reactivity, thermal stability, hygroscopicity, density, hardness, etc.

Thus it can be stated that different arrangement and conformation change of molecules in the crystal structure lead to differences in properties as follows: (*i*) mechanical (hardness, compactness, etc.); (*ii*) surface (surface energy, interfacial tension, etc.); (*iii*) kinetic (dissolution values, stability, etc.); (*iv*) spectroscopic (electronic, vibrational state transition, etc.); (*v*) thermodynamic (melting point, sublimation, enthalpy, entropy, etc.); (*vi*) packing properties (molar volume a density, hygroscopicity, etc.) (Brittain, 2009).

## **2.3. Multi-component solids**

Usually solid crystal substances can exist as mono-component or multi-component. Monocomponent systems contain, as evident from the name, only one component, and multicomponent systems consist of two or more components. In pharmaceutics this is a crystal composed of an API and an additional molecule that determines the type of the multicomponent system, pseudopolymorph. As illustrated in Fig. 1, the additional molecule of hydrates is water; of solvates, a solvent; of salts, an ionizable component; and of co-crystals, a co-crystallization partner (Stahly, 2007; Kratochvíl, 2010). All these systems can be polymorphic (see Fig. 1 and Fig. 2). Due to the breadth of the topic and the focus of this paper on preparation of co-crystals, only co-crystals will be described in more details.

## **3. Co-crystals**

The term "co-crystal" (also written as cocrystal) originates from "a composite crystal" (Desiraju, 2003). The term and the definition of co-crystal is a subject of topical debate. In principle this is a multi-component system of host and guest type; both components are solid in pure state and under ambient conditions. There are a lot of definitions; for example, Stahly defines co-crystals as molecular complexes that contain two or more different molecules in the same crystal structure (Stahly, 2007). Bhogala and Nangia define co-crystals as multi-component solid-state assemblies of two or more compounds held together by any type or combination of intermolecular interactions (Bhogala & Nangia, 2008). Childs and Hardcastle define co-crystals as a crystalline material made up of two or more components, usually in a stoichiometric ratio, each component being an atom, ionic compound, or molecule (Childs & Hardcastle, 2007). The simplest definition of co-crystals was proposed by Bond: "synonym for multi-component molecular crystal" (Bond, 2007). Also the definition of Aakeröy and Salmon can be recognized. They describe co-crystal as stoichiometric structurally homogeneous multi-component crystalline material of host-guest type that contains two or more neutral building blocks (discrete neutral molecular species that are solids at ambient conditions) that are present in definite amounts whereas all solids containing ions, including complex transition-metal ions, are excluded) (Aakeröy & Salmon, 2005). Jones defines co-crystal as a crystalline complex of two or more neutral molecular constituents bound together in the crystal structure through non-covalent interactions, often including hydrogen bonding (Jones et al., 2006) and Zaworotko says that co-crystals are formed between a molecular or ionic API and a co-crystal former that is a solid under ambient conditions (Vishweshwar et al., 2006a).

The host is an API, and the guest is a co-crystallization partner (an excipient or another API). Note that according to the definition by Aakeröy and Salmon co-crystals are different from solvates by the fact that the host and the guest of co-crystals are in the solid phase, while solvates contain both a solid phase and a solvent (or its residual), *i.e.* a liquid phase. However, there is still the problem of a boundary between the salt and the co-crystal, because according to the last mentioned definition of Aakeröy and Salmon, ions as co-crystal components are excluded, so on Fig. 3 only the first example of two is a co-crystal according to the mentioned definition. The borderline between salts and co-crystals is blurred and can be distinguished by the location of the proton between an acid and a base. This state can be denoted as salt–co-crystal continuum (Childs et al., 2007).

Several types of co-crystals can be distinguished: (*i*) "simple" co-crystals; (*ii*) solvated (hydrated) co-crystals – the co-crystal contains a component that is liquid at ambient temperature; (*iii*) salt co-crystals – the host is an ionized form; (*iv*) solvated salt co-crystals; (*v*) polymorphs of all previous types of co-crystals.

## **3.1. Properties of co-crystals**

86 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

change from agonist to antagonist or vice versa.

**2.3. Multi-component solids**

**3. Co-crystals**

Though the biological effect of an API is induced by interaction of the drug molecule with a target receptor when cell natural chemistry is influenced primarily by conformation changes, it is important in what solid phase the drug is administered to the patient. That means that not only the molecular but also the crystalline structure of an API is essential. The crystalline structure influences not only chemical and physical stability but also the drug dissolution rate and so can affect markedly the drug bioavailability. On the other hand, an amorphous form may have a considerably higher dissolution rate than the most stable polymorph. Great differences in the solubility and the dissolution rate of polymorphs can be a reason of significant differences in their distribution in the organism. Low plasma concentration can theoretically cause incomplete occupation of respective membrane receptors at the site of action (they are blocked by a substrate), or biological activity can

However, the dissolution rate is not the only important parameter of the difference between polymorphs. There can be also differences in crystal size and shape that influence milling, tabletting, filtration, looseness and other important technological parameters. There are also differences in chemical reactivity, thermal stability, hygroscopicity, density, hardness, etc.

Thus it can be stated that different arrangement and conformation change of molecules in the crystal structure lead to differences in properties as follows: (*i*) mechanical (hardness, compactness, etc.); (*ii*) surface (surface energy, interfacial tension, etc.); (*iii*) kinetic (dissolution values, stability, etc.); (*iv*) spectroscopic (electronic, vibrational state transition, etc.); (*v*) thermodynamic (melting point, sublimation, enthalpy, entropy, etc.); (*vi*) packing

Usually solid crystal substances can exist as mono-component or multi-component. Monocomponent systems contain, as evident from the name, only one component, and multicomponent systems consist of two or more components. In pharmaceutics this is a crystal composed of an API and an additional molecule that determines the type of the multicomponent system, pseudopolymorph. As illustrated in Fig. 1, the additional molecule of hydrates is water; of solvates, a solvent; of salts, an ionizable component; and of co-crystals, a co-crystallization partner (Stahly, 2007; Kratochvíl, 2010). All these systems can be polymorphic (see Fig. 1 and Fig. 2). Due to the breadth of the topic and the focus of this

paper on preparation of co-crystals, only co-crystals will be described in more details.

The term "co-crystal" (also written as cocrystal) originates from "a composite crystal" (Desiraju, 2003). The term and the definition of co-crystal is a subject of topical debate. In principle this is a multi-component system of host and guest type; both components are solid in pure state and under ambient conditions. There are a lot of definitions; for example, Stahly defines co-crystals as molecular complexes that contain two or more different

properties (molar volume a density, hygroscopicity, etc.) (Brittain, 2009).

Pharmaceutical co-crystals, that is, co-crystals that are formed between an API and pharmaceutically acceptable (GRAS) compounds (co-crystal formers, counterions) that are solid under ambient conditions, represent a new paradigm in API formulation that might address important intellectual and physical property issues in the context of drug development and delivery.

Co-crystals can be understood as "addition compounds" or "organic molecular compounds". They are attractive to the pharmaceutical industry, because they offer opportunities to modify the chemical and/or physical properties of an API without the need to make or break covalent

bonds (see Fig. 4). The term "non-covalent derivatization" was coined for this approach. Cocrystals of an API with excipients become very important as a tool to tune solubility and absorption. The application of co-crystal technologies has only recently been recognised as a way to enhance solubility, stability and the intellectual property position with respect to development of APIs (Vishweshwar et al., 2006b; Sekhon, 2009; Schultheiss & Newman, 2009).

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 89

**Figure 4.** Frequency of occurrence of organic molecular co-crystals in the Cambridge Structural Database from 1988 to 2007. For the purposes of this graph, co-crystals are distinct from solvates, hydrates and simple salts. (Ref. Childs & Zaworotko, 2009; reprinted with permission from Childs S.L., Zaworotko M.J. The reemergence of cocrystals: The crystal clear writing is on the wall introduction to virtual special issue on pharmaceutical cocrystals. Crystal Growth&Design 2009, 9(10): 4208-4211.

Scientists showed that modifying the physical properties of a pharmaceutical compound through pharmaceutical co-crystal formation improved the performance of a drug known to have poor solubility (Vishweshwar et al., 2006a). Pharmaceutical co-crystallization is a reliable method to modify physical and technical properties of drugs such as solubility increase/decrease, change of dissolution rate, stability, hygroscopicity and compressibility without alternating their pharmacological behaviour (Ranganathan, 1999; Fleischman et al., 2003; Almarsson & Zaworotko, 2004; Childs et al., 2004; Peterson et al.; 2006; Hickey et al.,

The use of co-crystals in drug design and delivery and as functional materials with potential applications as pharmaceuticals has recently attracted considerable interest (Fleischman et al., 2003; Jones et al., 2006; McNamara et al., 2006; Peterson et al., 2006; Vishweshwar et al., 2006a).  Co-crystals of antitubercular drugs with dicarboxylic acids were reported using carboxylic acid-pyridine synthon as a reliable tool (Vishweshwar et al., 2003). The co-crystal of piracetam-L-tartaric acid showed improved hygroscopic properties

Copyright 2009 American Chemical Society.)

2007; Žegarać et al., 2007; Schultheiss & Newman, 2009).

Co-crystallization with pharmaceutical excipients does not affect pharmacological activity of an API but can improve physical properties, such as solubility, hygroscopicity and compaction behaviour (Aakeröy et al., 2007; Rodríguez-Hornedo et al., 2007; Trask, 2007; Sun & Hou, 2008; Zaworotko, 2008). Co-crystals with the same active pharmaceutical ingredient will have strikingly different pharmaceutical properties (melting point, solubility, dissolution, bioavailability, moisture uptake, chemical stability, etc.), depending on the nature of the second component. Some of co-crystals formed had higher and some lower melting points as compared to their pure components, for example, succinic acid (Mp=135.3), urea (Mp=188.9), co-crystal of succinic acid-urea (Mp=149.9) (Walsh et al., 2003).

**Figure 3.** Possible multi-component systems: co-crystals, salt co-crystals and derived multi-component solids. (Ref. Schultheiss & Newman, 2009; reprinted and adapted with permission from Schultheiss N., Newman A. Pharmaceutical co-crystals and their physicochemical properties. Crystal Growth & Design 2009, 9(6): 2950–2967. Copyright 2009 American Chemical Society.)

bonds (see Fig. 4). The term "non-covalent derivatization" was coined for this approach. Cocrystals of an API with excipients become very important as a tool to tune solubility and absorption. The application of co-crystal technologies has only recently been recognised as a way to enhance solubility, stability and the intellectual property position with respect to development of APIs (Vishweshwar et al., 2006b; Sekhon, 2009; Schultheiss & Newman, 2009). Co-crystallization with pharmaceutical excipients does not affect pharmacological activity of an API but can improve physical properties, such as solubility, hygroscopicity and compaction behaviour (Aakeröy et al., 2007; Rodríguez-Hornedo et al., 2007; Trask, 2007; Sun & Hou, 2008; Zaworotko, 2008). Co-crystals with the same active pharmaceutical ingredient will have strikingly different pharmaceutical properties (melting point, solubility, dissolution, bioavailability, moisture uptake, chemical stability, etc.), depending on the nature of the second component. Some of co-crystals formed had higher and some lower melting points as compared to their pure components, for example, succinic acid (Mp=135.3),

urea (Mp=188.9), co-crystal of succinic acid-urea (Mp=149.9) (Walsh et al., 2003).

**Figure 3.** Possible multi-component systems: co-crystals, salt co-crystals and derived multi-component solids. (Ref. Schultheiss & Newman, 2009; reprinted and adapted with permission from Schultheiss N., Newman A. Pharmaceutical co-crystals and their physicochemical properties. Crystal Growth & Design

2009, 9(6): 2950–2967. Copyright 2009 American Chemical Society.)

**Figure 4.** Frequency of occurrence of organic molecular co-crystals in the Cambridge Structural Database from 1988 to 2007. For the purposes of this graph, co-crystals are distinct from solvates, hydrates and simple salts. (Ref. Childs & Zaworotko, 2009; reprinted with permission from Childs S.L., Zaworotko M.J. The reemergence of cocrystals: The crystal clear writing is on the wall introduction to virtual special issue on pharmaceutical cocrystals. Crystal Growth&Design 2009, 9(10): 4208-4211. Copyright 2009 American Chemical Society.)

Scientists showed that modifying the physical properties of a pharmaceutical compound through pharmaceutical co-crystal formation improved the performance of a drug known to have poor solubility (Vishweshwar et al., 2006a). Pharmaceutical co-crystallization is a reliable method to modify physical and technical properties of drugs such as solubility increase/decrease, change of dissolution rate, stability, hygroscopicity and compressibility without alternating their pharmacological behaviour (Ranganathan, 1999; Fleischman et al., 2003; Almarsson & Zaworotko, 2004; Childs et al., 2004; Peterson et al.; 2006; Hickey et al., 2007; Žegarać et al., 2007; Schultheiss & Newman, 2009).

The use of co-crystals in drug design and delivery and as functional materials with potential applications as pharmaceuticals has recently attracted considerable interest (Fleischman et al., 2003; Jones et al., 2006; McNamara et al., 2006; Peterson et al., 2006; Vishweshwar et al., 2006a).  Co-crystals of antitubercular drugs with dicarboxylic acids were reported using carboxylic acid-pyridine synthon as a reliable tool (Vishweshwar et al., 2003). The co-crystal of piracetam-L-tartaric acid showed improved hygroscopic properties (Viertelhaus et al., 2009). Co-crystal forming abilities of two anti-HIV drugs (lamivudine and zidovudine) were studied to investigate the general applicability (Bhatt et al., 2009). Trimer co-crystals of *cis*-itraconazole-succinic acid were prepared and characterized by the possibility of achieving the higher oral bioavailability normally observed for amorphous forms of water-insoluble drugs (Remenar et al., 2003). The novel pharmaceutical co-crystal norfloxacin saccharinate dihydrate and its co-crystal, norfloxacin saccharinate–saccharin dihydrate were reported (Velaga et al., 2008).

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 91

bonds, and (*iii*) the best proton donors and acceptors remaining after intramolecular hydrogen-bond formation form intermolecular hydrogen bonds with one another. In addition, the selectivity of hydrogen bonding in co-crystals was demonstrated by using

Based on the above mentioned facts co-crystal prediction includes the following steps: (*i*) determining whether a given set of two or more molecular components will undergo cocrystallization; (*ii*) identifying the primary intermolecular interactions, e.g., hydrogen-bond motifs that will exist within a particular co-crystal structure; and (*iii*) envisioning the overall

Design and preparation of pharmaceutical co-crystals is a multi-stage process that can be schematically described in the following steps: (*i*) selection and research of APIs; (*ii*) selection of co-crystal formers; (*iii*) empirical and theoretical guidance; (*iv*) co-crystal screening; (*v*) co-crystal characterisation; (*vi*) co-crystal performance (Miroshnyk et al., 2009;

O

R<sup>1</sup> O H

f

H O

H N

R3

R2 O R3 R2

O

R1

H O

d

H N

O

R2

R3

O

O

**Figure 5.** Possible formation of hydrogen bonds between synthons used in crystal engineering: acidacid (a), acid-amine (b), acid-amide (c), acid-imide (d), amide-amide (e), and alcohol-ether (f).

R3

A pharmaceutical co-crystal is a single-crystalline solid that incorporates two neutral molecules, one being an API and the other a co-crystal former (Vishweshwar et al., 2006). Co-crystal former may be an excipient or another drug (Rodríguez-Hornedo et al., 2007). Pharmaceutical co-crystal technology is used to identify and develop new proprietary forms of widely prescribed drugs and offers a chance to increase the number of forms of an API.

Crystalline forms can be generated by means of kinetically or thermodynamically controlled crystallization processes. Synthesis/processing of co-crystals can be accomplished via a number of methods, including slow solvent evaporation crystallization from solution, solvent-reduced (e.g. slurrying, solvent-drop grinding) and solvent-free (e.g. grinding, melt), high throughput crystallization and co-sublimation techniques (Shekunov & York,

packing arrangement in the resulting co-crystal structure (Trask, 2007).

Qiao et al., 2011).

 Synthesis of co-crystals**

## **3.2. Design of co-crystals**

Co-crystallization is a result of competing molecular associations between similar molecules, or homomers, and different molecules, or heteromers. Instead, both components (host and guest) utilise prominent intermolecular non-covalent interactions such as hydrogen bonding, van der Waals forces and π-π stacking interactions to combine and yield a uniform crystalline material. Hydrogen bonds are the basis of molecular recognition phenomena in pharmaceutical systems and are responsible for the generation of families of molecular networks with the same molecular components (single-component crystals and their polymorphs) or with different molecular components (multiple-component crystals or co-crystals) in the crystalline state (Jayasankar et al., 2006; Sekhon, 2009). The components in a co-crystal exist in simple definite stoichiometric ratios, e.g., 1:1, 1:2, 2:1, etc. Co-crystals have different crystal structures than the pure components, contain different intermolecular packing patterns and as such they often exhibit different physical properties than the pure components. Unlike salt formation, co-crystallisation does not rely on ionisation of the API and the counterion to make a solid. Co-crystals are an alternative to salts when these do not have the appropriate solid state properties or cannot be formed due to the absence of ionization sites in the API (Aakeröy & Salmon, 2005; Miroshnyk et al., 2009).

The formation of a salt or a co-crystal can be predicted from pKa value of an acid (A) and a base (B). Salt formation generally requires a difference of about 3 pKa units between the conjugate base and the conjugate acid (A) *i.e.* [pKa (B) – pKa (A) ≥ 3] (Etter, 1990; Whitesides & Wong, 2006; Sekhon, 2009). In cases when ΔpKa = pKa (B) – pKa (A) = 0-3, the transfer of proton is ambiguous, and we can talk about the salt–co-crystal continuum (Childs et al., 2007).

Co-crystals can be prepared from two molecules of any shape or size having complementary hydrogen bond functionalities. The ability of an API to form a co-crystal is dependent on a range of variables, including the types of co-former, the API co-former ratio, the solvents, the temperature, the pressure, the crystallization technique, etc. Common functional groups, such as carboxylic or amino acids, amides, alcohols and carbohydrates are typically found to interact with one another in co-crystals (see Fig. 5) (Miroshnyk et al., 2009, Sarma et al., 2011; Qiao et al., 2011). Etter has studied hydrogen bonds in co-crystals and uses them as design elements. The hydrogen bond general rules are the following: (*i*) all good proton donors and acceptors are used in hydrogen bonding, (*ii*) if six-membered ring intramolecular hydrogen bonds can form, they will usually do so in preference to forming intermolecular hydrogen bonds, and (*iii*) the best proton donors and acceptors remaining after intramolecular hydrogen-bond formation form intermolecular hydrogen bonds with one another. In addition, the selectivity of hydrogen bonding in co-crystals was demonstrated by using pyridines (Etter, 1991; Sarma et al., 2011).

Based on the above mentioned facts co-crystal prediction includes the following steps: (*i*) determining whether a given set of two or more molecular components will undergo cocrystallization; (*ii*) identifying the primary intermolecular interactions, e.g., hydrogen-bond motifs that will exist within a particular co-crystal structure; and (*iii*) envisioning the overall packing arrangement in the resulting co-crystal structure (Trask, 2007).

Design and preparation of pharmaceutical co-crystals is a multi-stage process that can be schematically described in the following steps: (*i*) selection and research of APIs; (*ii*) selection of co-crystal formers; (*iii*) empirical and theoretical guidance; (*iv*) co-crystal screening; (*v*) co-crystal characterisation; (*vi*) co-crystal performance (Miroshnyk et al., 2009; Qiao et al., 2011).

**Figure 5.** Possible formation of hydrogen bonds between synthons used in crystal engineering: acidacid (a), acid-amine (b), acid-amide (c), acid-imide (d), amide-amide (e), and alcohol-ether (f).

#### **3.3. Synthesis of co-crystals**

90 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

dihydrate were reported (Velaga et al., 2008).

**3.2. Design of co-crystals**

2007).

(Viertelhaus et al., 2009). Co-crystal forming abilities of two anti-HIV drugs (lamivudine and zidovudine) were studied to investigate the general applicability (Bhatt et al., 2009). Trimer co-crystals of *cis*-itraconazole-succinic acid were prepared and characterized by the possibility of achieving the higher oral bioavailability normally observed for amorphous forms of water-insoluble drugs (Remenar et al., 2003). The novel pharmaceutical co-crystal norfloxacin saccharinate dihydrate and its co-crystal, norfloxacin saccharinate–saccharin

Co-crystallization is a result of competing molecular associations between similar molecules, or homomers, and different molecules, or heteromers. Instead, both components (host and guest) utilise prominent intermolecular non-covalent interactions such as hydrogen bonding, van der Waals forces and π-π stacking interactions to combine and yield a uniform crystalline material. Hydrogen bonds are the basis of molecular recognition phenomena in pharmaceutical systems and are responsible for the generation of families of molecular networks with the same molecular components (single-component crystals and their polymorphs) or with different molecular components (multiple-component crystals or co-crystals) in the crystalline state (Jayasankar et al., 2006; Sekhon, 2009). The components in a co-crystal exist in simple definite stoichiometric ratios, e.g., 1:1, 1:2, 2:1, etc. Co-crystals have different crystal structures than the pure components, contain different intermolecular packing patterns and as such they often exhibit different physical properties than the pure components. Unlike salt formation, co-crystallisation does not rely on ionisation of the API and the counterion to make a solid. Co-crystals are an alternative to salts when these do not have the appropriate solid state properties or cannot be formed due to the absence of

ionization sites in the API (Aakeröy & Salmon, 2005; Miroshnyk et al., 2009).

The formation of a salt or a co-crystal can be predicted from pKa value of an acid (A) and a base (B). Salt formation generally requires a difference of about 3 pKa units between the conjugate base and the conjugate acid (A) *i.e.* [pKa (B) – pKa (A) ≥ 3] (Etter, 1990; Whitesides & Wong, 2006; Sekhon, 2009). In cases when ΔpKa = pKa (B) – pKa (A) = 0-3, the transfer of proton is ambiguous, and we can talk about the salt–co-crystal continuum (Childs et al.,

Co-crystals can be prepared from two molecules of any shape or size having complementary hydrogen bond functionalities. The ability of an API to form a co-crystal is dependent on a range of variables, including the types of co-former, the API co-former ratio, the solvents, the temperature, the pressure, the crystallization technique, etc. Common functional groups, such as carboxylic or amino acids, amides, alcohols and carbohydrates are typically found to interact with one another in co-crystals (see Fig. 5) (Miroshnyk et al., 2009, Sarma et al., 2011; Qiao et al., 2011). Etter has studied hydrogen bonds in co-crystals and uses them as design elements. The hydrogen bond general rules are the following: (*i*) all good proton donors and acceptors are used in hydrogen bonding, (*ii*) if six-membered ring intramolecular hydrogen bonds can form, they will usually do so in preference to forming intermolecular hydrogen A pharmaceutical co-crystal is a single-crystalline solid that incorporates two neutral molecules, one being an API and the other a co-crystal former (Vishweshwar et al., 2006). Co-crystal former may be an excipient or another drug (Rodríguez-Hornedo et al., 2007). Pharmaceutical co-crystal technology is used to identify and develop new proprietary forms of widely prescribed drugs and offers a chance to increase the number of forms of an API.

Crystalline forms can be generated by means of kinetically or thermodynamically controlled crystallization processes. Synthesis/processing of co-crystals can be accomplished via a number of methods, including slow solvent evaporation crystallization from solution, solvent-reduced (e.g. slurrying, solvent-drop grinding) and solvent-free (e.g. grinding, melt), high throughput crystallization and co-sublimation techniques (Shekunov & York, 2000; Morissette et al., 2004; Trask & Jones, 2005; Trask et al., 2005a; Trask et al., 2005b; Trask et al., 2006; Stahly, 2007; Berry et al., 2008; Takata et al., 2008; Friščić & Jones, 2009; Schultheiss & Newman, 2009; Qiao et al., 2011). Typically co-crystals are prepared by slow solvent evaporation that is only viable if compatible solubility in a given solvent exists between the components comprising the potential co-crystal. The potential benefits, disadvantages and methods of preparation of co-crystals were reported (Blagden et al., 2007). Solvent drop grinding has been reported to be a cost-effective, green, and reliable method for discovery of new co-crystals as well as for preparation of existing co-crystals. A slurry crystallization technique was used in co-crystal screening of two non-ionizable pharmaceutical host compounds, stanolone and mestanolone, with 11 pharmaceutically acceptable guest acids, and the results demonstrated the importance not only of hydrogen bonding but also of geometric fit in co-crystal formation (Takata et al., 2008). In addition to these classical techniques of co-crystal synthesis, also more sophisticated methods can be mentioned such as electrochemical crystallization, gel crystallization, vapour-diffusion crystallization, cryogenic grinding (cryomilling), microporous membranes crystallization, supercritical fluids crystallization and sonocrystallization (Dahlin et al., 2011; Hsu et al., 2002; Forsythe et al., 2002; He & Lavernia, 2001; Di Profio et al., 2007; Tong et al., 2001; Ruecroft et al., 2005). The combinations and variations of the above techniques may be used to cause co-crystal formation (McMahon et al., 2007). It is evident that many of the reported co-crystals appear to be the result of serendipity, although several groups have successfully exploited crystal engineering principles for design and synthesis of co-crystals.

Investigation of Carbohydrates and Their Derivatives as Crystallization Modifiers 93

materials, as are the instruments working in the mid infrared region, due to different radiation frequencies used in the NIR spectrometry. Acquisition of NIR spectra requires several seconds and can be performed during manufacturing process. Therefore, the NIR spectroscopy is still more often applied as a tool of process analytical technology. In co-crystal analysis NIR spectroscopy is commonly used as a screening method of the first

The aim is not only to present the basic principles of both methods, but also to compare them. Raman spectroscopy and mid-infrared spectroscopy are both widely used in the pharmaceutical industry for the solid-state characterization because of their specificity. MIR region is the most important region in terms of analytical application. It is a region, where the majority of the so-called fundamental vibrations appear, *i.e.* the vibrational transitions from the basic to the first excited vibrational state. Vibrational spectroscopy provides key information about the structure of molecules. Positions and intensities of bands in a vibrational spectrum can be used to determine the structure of a molecule or to determine the chemical identity of a sample. With sufficient experience it is possible to identify chemical compounds or monitor intermolecular interactions by evaluating changes in positions and intensities of Raman bands which is extremely useful for the above mentioned purposes.

The mid-infrared and Raman spectra (with the exception of optical isomers) of a drug substance or any chemical compound are unique. Raman spectroscopy is a vibrational spectroscopy method which complements mainly the mid-IR spectroscopy. The intensity of bands in IR spectra is proportional to the dipole moment change occurring during the given type of the vibrational motion. Modes with a large change in the dipole moment having intensive bands in the IR spectra generally provide low-intensity bands in Raman spectra. Conversely, vibrations of non-polar functional groups provide intense bands in Raman spectra and weak bands in infrared spectra. (Zakrzewski & Zakrzewski, 2006; Pekárek & Jampílek, 2010). An important advantage of Raman spectroscopy over IR spectroscopy is the possibility to measure aqueous solutions. This advantage can be used in identification of

This technique covers a wide interval, which can be roughly defined, for example, by frequencies of 100 GHz and 3 THz, which corresponds to wavelengths between 3 and 0.1 mm, *i.e.* wave numbers from 3 to 100 cm-1. Hence THz region partially overlaps with far-infrared region. The characteristic frequency of 1 THz can be equivalently expressed in other spectroscopic units. The decisive factor for expansion of this method was the development of optical femtosecond lasers being an integral part of the most current laboratory and commercial THz spectrometers. Attractiveness of the THz field lies in the scale of the specific options for application of electromagnetic radiation of these frequencies. Application options in pharmaceutical analysis are mainly spectroscopy and imaging.

choice (Pekárek & Jampílek, 2010).

*4.1.2. MIR spectroscopy and Raman scattering*

APIs in aqueous solutions, emulsions or suspensions.

*4.1.3. THz spectroscopy*

## **4. Characterization of co-crystals**

Co-crystal characterisation is an important constituent part of co-crystal research. The basic techniques of analysis of co-crystals involve especially solid state analysis methods (Zakrzewski & Zakrzewski, 2006; Dohnal et al., 2010), *i.e.* vibration-rotation spectroscopy, solid state NMR, thermal analysis, microscopy techniques and X-ray diffraction. The most often used solid-state analytical techniques will be discussed below.

## **4.1. Spectroscopy of vibration-rotation transitions**

According to the used part of infrared (IR) spectra (energy), near infrared spectroscopy (NIR), middle infrared spectroscopy (MIR), Raman scattering and terahertz (THz) spectroscopy are distinguished.

## *4.1.1. NIR Spectroscopy*

Photons in NIR region have the highest energy and can therefore vibrationally excite molecules into even higher excited vibrational states than the first level, *i.e.* the second, the third and others. These transitions are called overtons. Absorption of radiation in the NIR region is usually based on higher energy transitions between vibrational levels of molecules, namely combination transitions and overtones and not fundamental transitions, which are dominant in mid infrared region (MIR). NIR spectrometers are not so demanding of applied materials, as are the instruments working in the mid infrared region, due to different radiation frequencies used in the NIR spectrometry. Acquisition of NIR spectra requires several seconds and can be performed during manufacturing process. Therefore, the NIR spectroscopy is still more often applied as a tool of process analytical technology. In co-crystal analysis NIR spectroscopy is commonly used as a screening method of the first choice (Pekárek & Jampílek, 2010).

### *4.1.2. MIR spectroscopy and Raman scattering*

92 Carbohydrates – Comprehensive Studies on Glycobiology and Glycotechnology

2000; Morissette et al., 2004; Trask & Jones, 2005; Trask et al., 2005a; Trask et al., 2005b; Trask et al., 2006; Stahly, 2007; Berry et al., 2008; Takata et al., 2008; Friščić & Jones, 2009; Schultheiss & Newman, 2009; Qiao et al., 2011). Typically co-crystals are prepared by slow solvent evaporation that is only viable if compatible solubility in a given solvent exists between the components comprising the potential co-crystal. The potential benefits, disadvantages and methods of preparation of co-crystals were reported (Blagden et al., 2007). Solvent drop grinding has been reported to be a cost-effective, green, and reliable method for discovery of new co-crystals as well as for preparation of existing co-crystals. A slurry crystallization technique was used in co-crystal screening of two non-ionizable pharmaceutical host compounds, stanolone and mestanolone, with 11 pharmaceutically acceptable guest acids, and the results demonstrated the importance not only of hydrogen bonding but also of geometric fit in co-crystal formation (Takata et al., 2008). In addition to these classical techniques of co-crystal synthesis, also more sophisticated methods can be mentioned such as electrochemical crystallization, gel crystallization, vapour-diffusion crystallization, cryogenic grinding (cryomilling), microporous membranes crystallization, supercritical fluids crystallization and sonocrystallization (Dahlin et al., 2011; Hsu et al., 2002; Forsythe et al., 2002; He & Lavernia, 2001; Di Profio et al., 2007; Tong et al., 2001; Ruecroft et al., 2005). The combinations and variations of the above techniques may be used to cause co-crystal formation (McMahon et al., 2007). It is evident that many of the reported co-crystals appear to be the result of serendipity, although several groups have successfully

exploited crystal engineering principles for design and synthesis of co-crystals.

often used solid-state analytical techniques will be discussed below.

**4.1. Spectroscopy of vibration-rotation transitions**

Co-crystal characterisation is an important constituent part of co-crystal research. The basic techniques of analysis of co-crystals involve especially solid state analysis methods (Zakrzewski & Zakrzewski, 2006; Dohnal et al., 2010), *i.e.* vibration-rotation spectroscopy, solid state NMR, thermal analysis, microscopy techniques and X-ray diffraction. The most

According to the used part of infrared (IR) spectra (energy), near infrared spectroscopy (NIR), middle infrared spectroscopy (MIR), Raman scattering and terahertz (THz)

Photons in NIR region have the highest energy and can therefore vibrationally excite molecules into even higher excited vibrational states than the first level, *i.e.* the second, the third and others. These transitions are called overtons. Absorption of radiation in the NIR region is usually based on higher energy transitions between vibrational levels of molecules, namely combination transitions and overtones and not fundamental transitions, which are dominant in mid infrared region (MIR). NIR spectrometers are not so demanding of applied

**4. Characterization of co-crystals**

spectroscopy are distinguished.

*4.1.1. NIR Spectroscopy*

The aim is not only to present the basic principles of both methods, but also to compare them. Raman spectroscopy and mid-infrared spectroscopy are both widely used in the pharmaceutical industry for the solid-state characterization because of their specificity. MIR region is the most important region in terms of analytical application. It is a region, where the majority of the so-called fundamental vibrations appear, *i.e.* the vibrational transitions from the basic to the first excited vibrational state. Vibrational spectroscopy provides key information about the structure of molecules. Positions and intensities of bands in a vibrational spectrum can be used to determine the structure of a molecule or to determine the chemical identity of a sample. With sufficient experience it is possible to identify chemical compounds or monitor intermolecular interactions by evaluating changes in positions and intensities of Raman bands which is extremely useful for the above mentioned purposes.

The mid-infrared and Raman spectra (with the exception of optical isomers) of a drug substance or any chemical compound are unique. Raman spectroscopy is a vibrational spectroscopy method which complements mainly the mid-IR spectroscopy. The intensity of bands in IR spectra is proportional to the dipole moment change occurring during the given type of the vibrational motion. Modes with a large change in the dipole moment having intensive bands in the IR spectra generally provide low-intensity bands in Raman spectra. Conversely, vibrations of non-polar functional groups provide intense bands in Raman spectra and weak bands in infrared spectra. (Zakrzewski & Zakrzewski, 2006; Pekárek & Jampílek, 2010). An important advantage of Raman spectroscopy over IR spectroscopy is the possibility to measure aqueous solutions. This advantage can be used in identification of APIs in aqueous solutions, emulsions or suspensions.

#### *4.1.3. THz spectroscopy*