*2.2.2 β-Defensins*

Human β-Defensin (HBD) 2, 3 of this subfamily have anti-*Acinetobacter* effects. HBD-2 is primarily produced by the epithelial lining of the respiratory and urinary tracts, and engaging is more effective on MDR clinical isolates than non-MDR isolates [167]. Longer than most of the natural AMPs, HBD-3 combined helix and β structure [147]. Even though the anti-*Acinetobacter* bactericidal effect is inhibited by exposure to human serum, it can kill all MDR and non-MDR *A. baumannii* clinical isolates at 4 μg/ml during 1.5 h in the serum-free environment. Thus, this peptide has the potential to be further studied for wounds infected by *A. baumannii* since it demonstrated wound-healing effects [97, 168].

### *2.2.3 α-Helical and antiparallel β-sheet defensins*

CL-defensin, belonging to the family of insect defensins, is predicted to have a characteristic N-terminal loop, an α-helix, and an antiparallel β-sheet, which was supported by circular dichroism spectroscopy [95]. In addition, this peptide induces pore formation in other Gram-positive bacteria and causes a small amount of membrane permeabilization in *A. baumannii* [95].

#### **2.3 Frog antimicrobial peptides**

#### *2.3.1 Magainin and pexiganan (its analog)*

The Magainin-1 and 2 are cationic, α-helical, and amphipathic AMPs ionophores isolated from the skin of the African clawed frog (*Xenopus laevis*) [168, 169]. The primary mechanism of antimicrobial activity is probably pore formation in outer and inner membranes, although the exact mechanism of action is not yet precise [98, 170]. Despite both have anti-*Acinetobacter* training, Magainin-2 is much stronger and able to inhibit the growth of sensitive and MDR strains of *A. baumannii* at 4.9–64 μg/ml, while reported as 256 μg/ml for Magainin-1 [86, 98]. Magainin-2 has some advantages, such as anticancer effect, stability at physiological salt

concentrations, lack of hemolytic activity, and toxicity for mammalian cells [98]. Furthermore, Magainin-2 can inhibit and eliminate the biofilm of *A. baumannii* [98]. Pexiganan AMP or MSI-78 is a synthetic analog of Magainin-2 with a potent and broad spectrum of action [171, 172]; it kills bacteria by forming toroidal pores in their cell membranes [172, 173]. Several studies have been performed on anti-*Acinetobacter* activity due to its being highly active against *Acinetobacter*. Pexiganan can inhibit the growth of MDR and sensitive clinical isolates of A*. baumannii* at a concentration of 1–8 μg/ml [100, 101, 174]. Jáskiewicz et al. studied the antimicrobial activity of eight peptides on *A. baumannii* ATCC 19606 reference strains. Among these, CAMEL and pexiganan showed potent antimicrobial and anti-biofilm activity [102].

#### *2.3.2 Brevinin-2 related peptide (B2RP)*

B2RP is an α-helical AMP isolated from the skin secretions of the mink frog *Rana septentrionalis* [175] and carpenter frog Rana virgatipes [176]. This peptide forms an α-helical structure adjacent to the target cell, resulting in the perturbation of the phospholipid bilayer that may lead to growth inhibition of bacterial death, and the application of this peptide for systemic use is limited due to the moderate toxicity for human red blood cells [177]. B2RP inhibited the growth of a susceptible strain of *A. baumannii* at 29 μg/ml concentration but inhibited the MDR isolates more efficiently at 7–13.9 μg/ml [103]. The analogs of these peptides (D4K, K16A, L18K) resulted in twofolds higher anti-*A. baumannii* activity and much lower hemolytic activity [103]. A study reported that the analog of B2RP with D4K substitution inhibited sensitive and colistin-resistant [103] and XDR isolates of *A. baumannii* [105].

#### *2.3.3 B2RP-ERa*

B2RP-ERa is a cationic AMP from the Brevinin family isolated from the skin of the Asian frog *Hylarana erythraea* [106, 178]. Shorter and with lower molecular weight, B2RP-ERa is structurally similar to B2RP. B2RP-ERa is an antiinflammatory peptide with no toxic effect on peripheral blood mononuclear cells [179] with low hemolytic activity [178], which could inhibit the growth of sensitive and drug-resistant *Acinetobacter* strains at 8–32 and 8–64 μg/ml, respectively [104, 106].

#### *2.3.4 Alyteserins*

Alyteserins are a class of cationic AMPs, which firstly reported their presence in norepinephrine-stimulated skin secretions of the midwife toad [180]. However, initial studies show that Alyteserin-1c has more significant inhibitory activity against Gram-negative bacteria, while Alyteserin-2a is more active against Grampositive bacteria [180], the anti-*A. baumannii* effects of these Alyteserins have already been proven [107, 108]. Alyteserin-1c is a cationic α-helical AMP with low hemolytic activity on human red blood cells firstly isolated from *Alytes obstetricans* [107, 180, 181]. The MIC and MBC against clinical isolates of MDR *A. baumannii* have been reported as 11.3–22.6 μg/ml [107]. Substitution of E4K on this AMP reduced the hemolytic activity, and enhanced the antimicrobial and cationic activity [107]. The analog [E4K] inhibits the growth of colistin-sensitive, colistinresistant, and XDR *A. baumannii* isolates at concentrations of 4–16 μg/ml, 4–16 μg/ ml [104], and 8–64 μg/ml, respectively [105]. Alyteserin-2a is also a tiny α-helical AMP that displays relatively weak antimicrobial and hemolytic activities. Despite its anti-*A. baumannii* potential was not high mainly, some structural changes resulted in lower toxicity against human erythrocytes and higher bactericidal effect (4–8 folds) against MDR isolates with MIC of 6.8–13.6 μg/ml [108].


**4.1 Saliva versus blood**

Intra-individual variability of the S/P ratio has been demonstrated for a number of drugs administered orally or intravenously [35]. Following the uptake of an orally applied substance in the intestine, arterial blood has a higher concentration than venous blood (positive arteriovenous difference). If the substance is completely absorbed but not significantly metabolised in a particular organ, the situation is reversed: the substance rediffuses from the cells into the blood (negative arteriovenous difference in the elimination phase). The various organs can be classified into two groups: those with a high blood flow (e.g. liver, kidney, brain, salivary glands), and those with a relatively low blood flow (e.g. skin, resting skeletal muscle, fat). In pharmacokinetics the first group of highly perfused organs is included in the central compartment, while the second group of less perfused organs belongs to the peripheral compartment. This must be taken into account when saliva concentrations are compared with blood concentrations from cubital veins in the peripheral compartment. In any case, salivary glands have a high blood flow, which means that the arteriovenous difference of freely diffusible substances is relatively small, with a ratio close to 1.0. Poor correlations between the two compartments have been documented in the literature, but neglect of the phenomenon described can only partly account for this [36].

Some comparative studies [37, 38] have found that drug concentrations in oral fluid cannot be used to accurately estimate drug concentrations in blood. A positive result in an oral fluid test may certainly confirm recent drug use, but it may only provide a semiquantitative assessment of the drug concentration in the blood (and only for some drugs). For psychiatric patients, oral fluid testing may be used as a non-invasive technique for evaluating substance use. In the case of drivers suspected of driving under the influence of drugs, oral fluid may be used for initial on-site screening tests (afterwards, it may be decided that a blood sample should be taken for forensic drug analysis).

**143**

**Table 3.**

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes*

amphetamines, benzodiazepines, cocaine, opiates, and delta9

Wille et al. (2009) [37] analysed blood and saliva samples by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography-mass spectrometry (LC–MS). Scatter plots and trend lines of the blood and oral fluid concentrations were created and the median, mean, range, and standard deviation (SD) of the oral fluid to blood (OF/B) ratios were calculated for different classes of drugs, including

nol. The ratios found in this study were in line with previously published results, but the range was wider. The OF/B ratios of drugs of abuse such as amphetamines, cocaine, and opiates were > 1 [amphetamine: median (range) 13 (0.5-182), methylenedioxyamphetamine: 4 (1-15), methylenedioxymethamphetamine: 6 (0.9-88), methamphetamine: 5 (2-23), cocaine: 22 (4-119), benzoylecgonine: 1 (0.2-11), morphine: 2 (0.8-6), and codeine: 10 (0.8-39)]. Unsurprisingly, the ratios for benzodiazepines were considerably lower: given their high protein binding and weak acidity, benzodiazepines typically have low oral fluid concentrations [diazepam: 0.02 (0.01-0.15), nordiazepam: 0.04 (0.01-0.23), oxazepam: 0.05 (0.03-0.14), and temazepam: 0.1 (0.06-0.54)]. For tetrahydrocannabinol, an OF/B ratio of 15 was found (range 0.01-569). The variability of the OF/B ratios in suspected drugged drivers was clearly mirrored in the data. Be that as it may, blood concentrations could not be reliably calculated from oral fluid concentrations, due to the wide

Gjerde H et al. (2010) [38] analysed 90 pairs of blood and oral fluid specimens from patients undergoing acute psychiatric treatment and 22 pairs of blood and oral fluid specimens from suspected drugged drivers, with the aim of comparing drug concentrations between the two biological matrices. The median oral fluid/blood drug concentration ratios for the most prevalent drugs were 0.036 diazepam, 0.027 nordiazepam, 7.1 amphetamine, 2.9 methamphetamine, 5.4 codeine, 1.9 morphine, and 4.7 tetrahydrocannabinol. For the six most prevalent drugs, the correlation coefficients between drug concentrations in oral fluid and blood ranged from 0.15 to 0.96. The results, therefore, showed large interindividual variations in drug concentration ratios between oral fluid and blood. This wide distribution of OF/B ratios indicated that drug concentrations in oral fluid may not be used to reliably

Such analytical variability could cause controversy in the judicial field, especially when the values obtained from saliva are only slightly higher than the cut-off levels

*Drug Average oral fluid to blood concentration ratio*

Barbiturates [39] 0.3 Ethanol [40] 1.07 Buprenorphine [41] 1 Codeine [42] 4 Methamphetamine [43] 2 MDMA [44] 7 Cocaine [45] 3 Diazepam [46] 0.01–0.02 Methadone [47] 1.6 Morphine [48] 0.8 Δ9- Tetrahydrocannabinol [49] 1.2

*DOI: http://dx.doi.org/10.5772/intechopen.95625*

range of ratios.

estimate drug concentrations in blood.

established by the law of various countries.

*Average oral fluid to blood concentration ratios for selected drugs.*

#### *Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes DOI: http://dx.doi.org/10.5772/intechopen.95625*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

use LC–MS as distinct from GC–MS to cater for the lower sample volumes and low detection limits, although a number of GC–MS techniques have exhibited adequate

Intra-individual variability of the S/P ratio has been demonstrated for a number of drugs administered orally or intravenously [35]. Following the uptake of an orally applied substance in the intestine, arterial blood has a higher concentration than venous blood (positive arteriovenous difference). If the substance is completely absorbed but not significantly metabolised in a particular organ, the situation is reversed: the substance rediffuses from the cells into the blood (negative arteriovenous difference in the elimination phase). The various organs can be classified into two groups: those with a high blood flow (e.g. liver, kidney, brain, salivary glands), and those with a relatively low blood flow (e.g. skin, resting skeletal muscle, fat). In pharmacokinetics the first group of highly perfused organs is included in the central compartment, while the second group of less perfused organs belongs to the peripheral compartment. This must be taken into account when saliva concentrations are compared with blood concentrations from cubital veins in the peripheral compartment. In any case, salivary glands have a high blood flow, which means that the arteriovenous difference of freely diffusible substances is relatively small, with a ratio close to 1.0. Poor correlations between the two compartments have been documented in the literature, but neglect of the phenomenon described can only

Some comparative studies [37, 38] have found that drug concentrations in oral fluid cannot be used to accurately estimate drug concentrations in blood. A positive result in an oral fluid test may certainly confirm recent drug use, but it may only provide a semiquantitative assessment of the drug concentration in the blood (and only for some drugs). For psychiatric patients, oral fluid testing may be used as a non-invasive technique for evaluating substance use. In the case of drivers suspected of driving under the influence of drugs, oral fluid may be used for initial on-site screening tests (afterwards, it may be decided that a blood sample should be

**142**

sensitivity [34].

**Figure 3.**

**4.1 Saliva versus blood**

partly account for this [36].

taken for forensic drug analysis).

**4. Medico-legal and toxicological issues**

*Ultra-high performance liquid chromatography.*

Wille et al. (2009) [37] analysed blood and saliva samples by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography-mass spectrometry (LC–MS). Scatter plots and trend lines of the blood and oral fluid concentrations were created and the median, mean, range, and standard deviation (SD) of the oral fluid to blood (OF/B) ratios were calculated for different classes of drugs, including amphetamines, benzodiazepines, cocaine, opiates, and delta9 -2 tetrahydrocannabinol. The ratios found in this study were in line with previously published results, but the range was wider. The OF/B ratios of drugs of abuse such as amphetamines, cocaine, and opiates were > 1 [amphetamine: median (range) 13 (0.5-182), methylenedioxyamphetamine: 4 (1-15), methylenedioxymethamphetamine: 6 (0.9-88), methamphetamine: 5 (2-23), cocaine: 22 (4-119), benzoylecgonine: 1 (0.2-11), morphine: 2 (0.8-6), and codeine: 10 (0.8-39)]. Unsurprisingly, the ratios for benzodiazepines were considerably lower: given their high protein binding and weak acidity, benzodiazepines typically have low oral fluid concentrations [diazepam: 0.02 (0.01-0.15), nordiazepam: 0.04 (0.01-0.23), oxazepam: 0.05 (0.03-0.14), and temazepam: 0.1 (0.06-0.54)]. For tetrahydrocannabinol, an OF/B ratio of 15 was found (range 0.01-569). The variability of the OF/B ratios in suspected drugged drivers was clearly mirrored in the data. Be that as it may, blood concentrations could not be reliably calculated from oral fluid concentrations, due to the wide range of ratios.

Gjerde H et al. (2010) [38] analysed 90 pairs of blood and oral fluid specimens from patients undergoing acute psychiatric treatment and 22 pairs of blood and oral fluid specimens from suspected drugged drivers, with the aim of comparing drug concentrations between the two biological matrices. The median oral fluid/blood drug concentration ratios for the most prevalent drugs were 0.036 diazepam, 0.027 nordiazepam, 7.1 amphetamine, 2.9 methamphetamine, 5.4 codeine, 1.9 morphine, and 4.7 tetrahydrocannabinol. For the six most prevalent drugs, the correlation coefficients between drug concentrations in oral fluid and blood ranged from 0.15 to 0.96. The results, therefore, showed large interindividual variations in drug concentration ratios between oral fluid and blood. This wide distribution of OF/B ratios indicated that drug concentrations in oral fluid may not be used to reliably estimate drug concentrations in blood.

Such analytical variability could cause controversy in the judicial field, especially when the values obtained from saliva are only slightly higher than the cut-off levels established by the law of various countries.

#### **Table 3.**

*Average oral fluid to blood concentration ratios for selected drugs.*

**Table 3** shows the average values of oral fluid to blood concentration ratios of selected drugs, based on various pharmacokinetic studies; the average ratios change depending on a number of factors, such as pH of oral fluid, protein binding and degree of contamination of the membranes in the oral cavity by recently consumed drug.

#### **4.2 Cut-off levels and analytical interpretation**

Interpretation of oral fluid drug test results depends to some extent on the purpose of testing. An employer may decide to implement a workplace drug testing programme primarily to detect drug abuse among employees (or even job applicants), especially regarding safety-sensitive positions or following a safety incident or accident. Random workplace testing could also serve as a deterrent to substance misuse in the general workforce. Drug treatment specialists carry out drug testing to foster drug abstinence and compliance with programme requirements. Numerous factors must be considered when interpreting drug test results. During this process, complex questions may be posed, depending on the nature of the drug-testing programme, and sometimes the answers sought go beyond reasonable scientific certainty. Patterns of metabolic disposition should be understood for each class of drugs. Of course, the interpretation of oral fluid tests requires knowledge of the unique features of this biological matrix, along with a thorough understanding of: the chemical and physiological factors that affect drug transfer into oral fluid; analytical factors; kinetic aspects of drug disposition; drug metabolic patterns; and potential risks of oral contamination and passive exposure. Generally speaking, it has been shown that oral fluid tests are most useful in the detection of recent drug use [50].

The use of a screening method can be justified in a forensic toxicology laboratory when there is a need to analyse a large number of samples in a short time and at low costs, with the advantages of high or total automation. Screening methods usually employ colorimetric, enzymatic, and immunochemical techniques. However, screening methods are characterised by low specificity (qualitative data) and high inaccuracy (quantitative data), particularly when several chemical species can be detected in the sample but not discriminated by the method (e.g. an unchanged compound and its metabolites, or various types of similar species of compounds). Given their intrinsic characteristics, these methods exclusively produce a presumptive result, that is to say the probable negativity (absence) or positivity (presence, better defined as "non-negativity") of the sample with respect to an analyte, or more often a class of substances, relative to a cut-off value set by the method. In any case, whatever the analytical specificity of the screening method, a positive result obtained through a single screening test cannot have forensic validity. It is therefore essential that this result is verified by a confirmatory analysis on a new sample rate.

The results of a quantitative analysis must be expressed in a uniform unit of measurement, so as to exclude interpretative doubts, directly comparable with any reference values (cut-off) and accepted by the International System of Units (SI). The uncertainty associated with the measurement performed must be indicated; at the same time, the comparison with threshold or reference values must take into account this uncertainty. **Tables 4** and **5** show the recommended cut-off levels of oral fluid tests according to EWDTS and SAMHSA guidelines.

Drugs and metabolites can be detected for a period of several hours to several days following drug exposure. Their concentrations in oral fluid are generally related to content in blood, but may also be present as residual drug in the oral cavity [11].

**145**

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes*

Morphine *Opiates (Morphine) 40 Opiates*

*Drug Screening Cut-off (ng/mL) Confirmation Cut-off*

Codeine *15* Norcodeina *2* 6-Acetylcodeine *2* Dihydrocodeine *15* 6-Monoacetylmorphine *2* METHADONE AND METABOLITES *L-Methadone, 50* 20

Cocaine *Cocaine + metabolites 30 8* Benzoylecgonine *8*

methamphetamine Amphetamines 40 *15* amphetamine *15* MDMA *15* MDA *15*

THC *THC 10 2*

*Recommended maximum screening and confirmation cut-off values for oral fluid tests in the workplace*

*(6-MAM) 4*

5 1

*(ng/mL)*

*15*

*AMPHETAMINE*: a synthetic substance related to natural sympathomimetic amines with central nervous stimulant activity. Amphetamine appears rapidly in oral fluid following administration and parallels plasma drug concentrations. Amphetamine is also produced as a metabolite of methamphetamine and from a variety of pharmaceutical products. A positive test result for amphetamine indicates amphetamine use; determination of d/l-isomer ratio should rule out the possibility

*METHAMPHETAMINE*: a synthetic sympathomimetic amine with central nervous stimulant activity similar to amphetamine but with more lasting effects. It is misused in numerous ways including smoking, snorting, injecting, and oral administration. Methamphetamine and amphetamine appear rapidly in plasma and oral fluid following administration. Determination of d/l-isomer ratio rules out the possibility that methamphetamine presence is due to the metabolism of another drug or use of an over-the-counter nasal inhaler. A positive test result for methamphetamine and amphetamine (methamphetamine < amphetamine) indicates

possible combined use of methamphetamine and amphetamine.

*METHYLENEDIOXYMETHAMPHETAMINE (MDMA)*: a synthetic, ring-substituted amphetamine derivative. N-demethylation of MDMA yields 3,4-methylenedioxyamphetamine (MDA), an active metabolite exhibiting similar pharmacological properties as the parent drug. O-demethylenation of MDMA and MDA produces 3,4-dihydroxymethamphetamine (HHMA) and

*DOI: http://dx.doi.org/10.5772/intechopen.95625*

OPIATES

BUPRENORPHINE AND METABOLITES

CANNABINOIDS

*according to EWDTS guidelines [51].*

**Table 4.**

COCAINE AND METABOLITE

AMPHETAMINE AND CONGENERS

of mystification with another drug.

In what follows, descriptions of the main drugs of abuse are given [50].

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes DOI: http://dx.doi.org/10.5772/intechopen.95625*

#### **Table 4.**

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

**4.2 Cut-off levels and analytical interpretation**

detection of recent drug use [50].

consumed drug.

**Table 3** shows the average values of oral fluid to blood concentration ratios of selected drugs, based on various pharmacokinetic studies; the average ratios change depending on a number of factors, such as pH of oral fluid, protein binding and degree of contamination of the membranes in the oral cavity by recently

Interpretation of oral fluid drug test results depends to some extent on the purpose of testing. An employer may decide to implement a workplace drug testing programme primarily to detect drug abuse among employees (or even job applicants), especially regarding safety-sensitive positions or following a safety incident or accident. Random workplace testing could also serve as a deterrent to substance misuse in the general workforce. Drug treatment specialists carry out drug testing to foster drug abstinence and compliance with programme requirements. Numerous factors must be considered when interpreting drug test results. During this process, complex questions may be posed, depending on the nature of the drug-testing programme, and sometimes the answers sought go beyond reasonable scientific certainty. Patterns of metabolic disposition should be understood for each class of drugs. Of course, the interpretation of oral fluid tests requires knowledge of the unique features of this biological matrix, along with a thorough understanding of: the chemical and physiological factors that affect drug transfer into oral fluid; analytical factors; kinetic aspects of drug disposition; drug metabolic patterns; and potential risks of oral contamination and passive exposure. Generally speaking, it has been shown that oral fluid tests are most useful in the

The use of a screening method can be justified in a forensic toxicology laboratory when there is a need to analyse a large number of samples in a short time and at low costs, with the advantages of high or total automation. Screening methods usually employ colorimetric, enzymatic, and immunochemical techniques. However, screening methods are characterised by low specificity (qualitative data) and high inaccuracy (quantitative data), particularly when several chemical species can be detected in the sample but not discriminated by the method (e.g. an unchanged compound and its metabolites, or various types of similar species of compounds). Given their intrinsic characteristics, these methods exclusively produce a presumptive result, that is to say the probable negativity (absence) or positivity (presence, better defined as "non-negativity") of the sample with respect to an analyte, or more often a class of substances, relative to a cut-off value set by the method. In any case, whatever the analytical specificity of the screening method, a positive result obtained through a single screening test cannot have forensic validity. It is therefore essential that this result is verified by a confirmatory analysis on a new sample rate. The results of a quantitative analysis must be expressed in a uniform unit of measurement, so as to exclude interpretative doubts, directly comparable with any reference values (cut-off) and accepted by the International System of Units (SI). The uncertainty associated with the measurement performed must be indicated; at the same time, the comparison with threshold or reference values must take into account this uncertainty. **Tables 4** and **5** show the recommended cut-off levels of

oral fluid tests according to EWDTS and SAMHSA guidelines.

Drugs and metabolites can be detected for a period of several hours to several days following drug exposure. Their concentrations in oral fluid are generally related to content in blood, but may also be present as residual drug in the oral

In what follows, descriptions of the main drugs of abuse are given [50].

**144**

cavity [11].

*Recommended maximum screening and confirmation cut-off values for oral fluid tests in the workplace according to EWDTS guidelines [51].*

*AMPHETAMINE*: a synthetic substance related to natural sympathomimetic amines with central nervous stimulant activity. Amphetamine appears rapidly in oral fluid following administration and parallels plasma drug concentrations. Amphetamine is also produced as a metabolite of methamphetamine and from a variety of pharmaceutical products. A positive test result for amphetamine indicates amphetamine use; determination of d/l-isomer ratio should rule out the possibility of mystification with another drug.

*METHAMPHETAMINE*: a synthetic sympathomimetic amine with central nervous stimulant activity similar to amphetamine but with more lasting effects. It is misused in numerous ways including smoking, snorting, injecting, and oral administration. Methamphetamine and amphetamine appear rapidly in plasma and oral fluid following administration. Determination of d/l-isomer ratio rules out the possibility that methamphetamine presence is due to the metabolism of another drug or use of an over-the-counter nasal inhaler. A positive test result for methamphetamine and amphetamine (methamphetamine < amphetamine) indicates possible combined use of methamphetamine and amphetamine.

*METHYLENEDIOXYMETHAMPHETAMINE (MDMA)*: a synthetic, ring-substituted amphetamine derivative. N-demethylation of MDMA yields 3,4-methylenedioxyamphetamine (MDA), an active metabolite exhibiting similar pharmacological properties as the parent drug. O-demethylenation of MDMA and MDA produces 3,4-dihydroxymethamphetamine (HHMA) and

**Table 5.**

**147**

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes*

3,4-dihydroxyamphetamine (HHA), respectively. MDMA is typically administered orally and reaches maximal blood concentrations in approximately 2 hours. Oral fluid concentrations of MDMA are highly correlated with plasma MDMA. Oral fluid concentrations of MDMA are an order of magnitude higher than in plasma; this is attributed to the high pKa of MDMA and low plasma–protein binding. A positive test result for MDMA (no MDA) indicates illicit MDMA use; a positive test result for MDMA and MDA suggests illicit MDMA use (presence of MDA probably due to metabolism of MDMA to MDA but if MDA ≥ MDMA, a combined use of

*3,4-METHYLENEDIOXYAMPHETAMINE (MDA)*: a synthetic, ring-substituted amphetamine derivative. MDA has been reported to appear in oral fluid following the administration of MDMA in concentrations representing approximately 4–5% of MDMA. Possible sources of MDA: illicit MDA, metabolite of illicit MDMA, metabolite of illicit MDEA. However, the confirmed presence of HHA and/or HMA

*3,4-METHYLENEDIOXYETHYLAMPHETAMINE (MDEA)*: a synthetic analogue which is generated when an ethyl group is substituted for the methyl group of MDMA. MDEA is metabolised by O-demethylenation and by N-dealkylation of the ethyl-group. The major metabolite is formed by O-demethylenation to yield N-ethyl-4-hydroxy-3-methoxyamphetamine (HME); N-dealkylation leads to the formation of the active metabolite MDA. A positive test result for MDEA without MDA means illicit MDEA use, otherwise (in co-presence with MDA) combined use

*DELTA-9-TETRAHYDROCANNABINOL (THC)*: a naturally occurring psychoactive constituent of *Cannabis sativa*. THC appears rapidly in plasma following the smoking of cannabis products and is found in oral fluid following smoked and oral ingestion. According to several studies, THC is more highly present in oral fluid than blood, primarily as a result of deposition in the oral cavity. THC tends to

*COCAINE*: a natural stimulant compound made from the leaves of the coca plant. Cocaine has a short half-life (approximately 1 hour) and is rapidly hydrolysed by hepatic esterases to benzoylecgonine (BZE) and ecognine methyl ester (EME). Cocaine and its metabolites appear rapidly in oral fluid following all routes of administration. Cocaine concentrations decrease rapidly within approximately 1 hour; thereafter, oral fluid concentrations appear to decline in parallel with concentrations of the drug in the blood.  Heroin is most commonly administered intravenously and by other parenteral routes, but may also be smoked. Heroin and 6-acetylmorphine appear in oral fluid within 2 minutes of administration. Drug and metabolite concentrations in oral fluid are generally similar to blood concentrations following intravenous administration, but may be substantially higher than blood when smoked. Elevated drug and metabolite concentrations following smoking are probably a consequence of residual drug deposited in the oral cavity. Thirty to sixty minutes after heroin is smoked, concentrations in oral fluid diminish considerably and begin to reflect blood concentrations. If 6-acetylmorphine and morphine are

*MORPHINE*: a natural opiate alkaloid isolated from the plant *Papaver somniferum*; it is also a metabolite of heroin and codeine. Following parenteral administration, morphine appears rapidly in saliva. Cone [48] reported an approximate

detected, the use of heroin (and not morphine) can be confirmed.

*DOI: http://dx.doi.org/10.5772/intechopen.95625*

illicit MDMA and illicit MDA is admissible).

or an initiated metabolism of MDEA.

48 hours for daily users.

in oral fluid would be useful to substantiate MDA use.

decline in a similar manner to plasma concentrations.

*Cut-off levels of oral fluid testing according to SAMHSA oral fluid guidelines [11] (effective January 1, 2020).*

#### *Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes DOI: http://dx.doi.org/10.5772/intechopen.95625*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

**146**

*Initial Test Analyte*

THC (Cannabis)

Cocaine/Benzoylecgonine

Codeine/Morphine

Hydrocodone/Hydromorphone

Oxycodone/Oxymorphone

6-Acetylmorphine (heroin)

Phencyclidine (PCP)

Amphetamine/Methamphetamine

Methylenedioxymethamphatime (MDMA)

Methylenedioxyamphetamine (MDA)

**Table 5.**

*Cut-off levels of oral fluid testing according to SAMHSA oral fluid guidelines [11] (effective January 1, 2020).*

50

MDMA MDA

*Screening Cut-off (ng/mL)*

4 15 30 30 30 4 10 50

6-Acetylmorphine

Phencyclidine Amphetamine/Methamphetamine

*Confirmatory Test Analyte*

THC Cocaine/Benzoylecgonine

Codeine Morphine Hydrocodone/Hydromorphone

Oxycodone/Oxymorphone

*Confirmatory Test Cut-off (ng/mL)*

2

8

8

15

15

15

15

15

15

2

10

25

25

25

25

3,4-dihydroxyamphetamine (HHA), respectively. MDMA is typically administered orally and reaches maximal blood concentrations in approximately 2 hours. Oral fluid concentrations of MDMA are highly correlated with plasma MDMA. Oral fluid concentrations of MDMA are an order of magnitude higher than in plasma; this is attributed to the high pKa of MDMA and low plasma–protein binding. A positive test result for MDMA (no MDA) indicates illicit MDMA use; a positive test result for MDMA and MDA suggests illicit MDMA use (presence of MDA probably due to metabolism of MDMA to MDA but if MDA ≥ MDMA, a combined use of illicit MDMA and illicit MDA is admissible).

*3,4-METHYLENEDIOXYAMPHETAMINE (MDA)*: a synthetic, ring-substituted amphetamine derivative. MDA has been reported to appear in oral fluid following the administration of MDMA in concentrations representing approximately 4–5% of MDMA. Possible sources of MDA: illicit MDA, metabolite of illicit MDMA, metabolite of illicit MDEA. However, the confirmed presence of HHA and/or HMA in oral fluid would be useful to substantiate MDA use.

*3,4-METHYLENEDIOXYETHYLAMPHETAMINE (MDEA)*: a synthetic analogue which is generated when an ethyl group is substituted for the methyl group of MDMA. MDEA is metabolised by O-demethylenation and by N-dealkylation of the ethyl-group. The major metabolite is formed by O-demethylenation to yield N-ethyl-4-hydroxy-3-methoxyamphetamine (HME); N-dealkylation leads to the formation of the active metabolite MDA. A positive test result for MDEA without MDA means illicit MDEA use, otherwise (in co-presence with MDA) combined use or an initiated metabolism of MDEA.

*DELTA-9-TETRAHYDROCANNABINOL (THC)*: a naturally occurring psychoactive constituent of *Cannabis sativa*. THC appears rapidly in plasma following the smoking of cannabis products and is found in oral fluid following smoked and oral ingestion. According to several studies, THC is more highly present in oral fluid than blood, primarily as a result of deposition in the oral cavity. THC tends to decline in a similar manner to plasma concentrations.

*COCAINE*: a natural stimulant compound made from the leaves of the coca plant. Cocaine has a short half-life (approximately 1 hour) and is rapidly hydrolysed by hepatic esterases to benzoylecgonine (BZE) and ecognine methyl ester (EME). Cocaine and its metabolites appear rapidly in oral fluid following all routes of administration. Cocaine concentrations decrease rapidly within approximately 1 hour; thereafter, oral fluid concentrations appear to decline in parallel with concentrations of the drug in the blood.

*HEROIN*: a semisynthetic opioid, diacetyl derivative of morphine prepared from opium for the illegal drug trade. Heroin is most commonly administered intravenously and by other parenteral routes, but may also be smoked. Heroin and 6-acetylmorphine appear in oral fluid within 2 minutes of administration. Drug and metabolite concentrations in oral fluid are generally similar to blood concentrations following intravenous administration, but may be substantially higher than blood when smoked. Elevated drug and metabolite concentrations following smoking are probably a consequence of residual drug deposited in the oral cavity. Thirty to sixty minutes after heroin is smoked, concentrations in oral fluid diminish considerably and begin to reflect blood concentrations. If 6-acetylmorphine and morphine are detected, the use of heroin (and not morphine) can be confirmed.

*MORPHINE*: a natural opiate alkaloid isolated from the plant *Papaver somniferum*; it is also a metabolite of heroin and codeine. Following parenteral administration, morphine appears rapidly in saliva. Cone [48] reported an approximate

45-minute delay in equilibration of morphine concentrations in saliva compared to plasma following intramuscular administration of 10- and 20-mg doses; thereafter, saliva concentrations paralleled plasma concentrations. Morphine can be detected in oral fluid following intravenous administration, the smoking of heroin and poppy seed ingestion. Positive tests for morphine and codeine (with higher codeine concentration) implies codeine use.

*CODEINE*: a naturally occurring phenanthrene alkaloid and opioid agonist. It appears to be most commonly taken orally. While it is not a metabolite of morphine, it is metabolised by oxidation to morphine and norcodeine and by conjugation. Kim et al. [52] demonstrated that following oral administration of 60 and 120 mg, codeine appeared in oral fluid within an hour and reached maximum concentration in approximately 1.6–1.7 hours. Concentrations in oral fluid correlated significantly with plasma concentration and were three to four times higher in oral fluid than plasma. Codeine could be detected in oral fluid for approximately 21 and 7 hours at cut-off concentrations of 2.5 and 40 ng/mL, respectively. Following intramuscular codeine of 60 and 120 mg, codeine appeared rapidly in oral fluid and reached maximal concentrations in 0.5–0.75 hours. A positive test result for codeine and morphine generally indicates codeine use.

*METHADONE*: a synthetic opioid used widely as an analgesic as well as in maintenance therapy for persons with opioid dependency. Methadone undergoes extensive metabolism in the liver to form cyclic metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrrolidine (EMDP), and other minor metabolites. Methadone and EDDP appear rapidly in oral fluid and correlate with plasma concentrations. Therefore, the confirmed presence of oxidative metabolites such as EDDP and EMDP in oral fluid would be useful to substantiate use.

*BUPRENORPHINE*: an orally available, semisynthetic opioid analgesic, used as a pain reliever and in the management of opioid dependence. Following sublingual administration, buprenorphine reaches maximal plasma concentrations in 1.3–1.6 hours. Its main metabolite is norbuprenorphine. Cone [48] reported measurements of buprenorphine in saliva following intramuscular and sublingual administration of single doses of buprenorphine. Drug concentrations in saliva were substantially lower than plasma at all times following intramuscular administration and were substantially higher following sublingual administration. The low S/P ratio following intramuscular administration is probably due to the high fraction of drug that is protein-bound in plasma. Close correspondence between saliva and plasma buprenorphine concentrations was observed in subjects who administered buprenorphine sublingually on a daily or every-other-day basis. If the oral fluid test reveals buprenorphine ≤ norbuprenorphine, this suggests chronic buprenorphine use.

#### **4.3 Quality assurance of the analysis**

Drug testing laboratories must implement a quality management system that includes all aspects of the testing process, such as sample reception, chain of custody, safety and reporting of results, screening and confirmation tests, certification of calibrators and controls, and validation of analytical procedures.

Hence, the laboratory should remain constantly updated on the evolution of analytical techniques, whether for finding new drugs or responding to requests for investigations concerning narcotic and pharmacological drugs. A highly qualified analytical chemical-toxicological laboratory depends on ISO/IEC 17025 [53] accreditation standards and procedures to demonstrate that it operates competently and generates valid results. These standards are agreed by experts the world over,

**149**

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes*

thus promoting confidence in the work of accredited laboratories and other bodies, on national and international levels, and facilitating cooperation between them. With ISO certification, results are more widely accepted between countries without

Of course, the benefits of advanced equipment in the chemical-toxicological laboratory go hand in hand with the expertise of qualified personnel with specific competence and adequate scientific training (not restricted to the analytical

chemical field). Only through this combination (state-of-the-art tools plus qualified personnel) will it be possible to develop new analytical methodologies in the field of toxicological-forensic analysis and respond to administrative, criminal and social

The validation of analytical methods includes procedures designed to establish that a particular method, used for the identification and/or quantification of an analyte in a given biological matrix, is reliable and reproducible. It is a question of demonstrating that the performance characteristics of the method meet all the requirements for its intended purpose and application. Any analysis methodology used routinely by the laboratory must be previously validated according to interna-

For the most commonly used screening tests, validation procedures are not usually necessary as the method is validated by the manufacturer. In any case, the analysis kit includes calibrators and controls which are to be inserted into each batch of samples to be analysed in order to verify the accuracy and precision of the analyses (according to predetermined target values). In the event that changes are introduced which deviate from the manufacturer's instructions (for example, the biological matrix used is not the one indicated by the manufacturer, variation of the quantification limit, etc.) the laboratory must carry out a complete validation of the method/modified kit. It is best to fully respect the instructions provided by the manufacturer in the use of a kit, or in any case modifications should only be carried

The analysis methodology can be used routinely by the laboratory if the calculated validation parameters fall within the limits established by the relevant inter-

The use of a good internal quality programme guarantees the reliability of the analytical results and avoids any random errors that may occur in the analytical and/or pre- or post-analytical phase that may affect the accuracy of the result. The laboratory must participate in appropriate external quality assessment programmes. Analytical performances outside the criteria established by the External Quality Assessment (EQA) programme must be promptly corrected. The choice of one programme over another must be made on the basis of the best scientific evidence obtainable. Participation may concern the identification of classes of substances or individual substances and quantification in the case of confirmatory analyses according to the legal cut-offs or established by the management body of the programme. In the case of screening tests, the expression of the results is generally in terms of "positive" or "negative". In the case of confirmatory analysis, it is necessary to provide not only qualitative but also quantitative data, namely the concentration detected according to a given calibration curve for the analyte identified in the saliva sample. The results of participation in the EQA are useful for the laboratory director and staff in helping to gauge the performance of the laboratory. In the event of errors, it is important to identify the causes and implement corrective

the need for further testing, consequently improving international trade.

*4.3.1 Validation of an analytical method for the detection of drugs in saliva*

out in cases where it is not possible to use other methods.

*DOI: http://dx.doi.org/10.5772/intechopen.95625*

needs imposed by the legal system.

tionally agreed procedures [17].

national directives [54].

actions that prevent them from recurring.

#### *Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes DOI: http://dx.doi.org/10.5772/intechopen.95625*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

concentration) implies codeine use.

morphine generally indicates codeine use.

useful to substantiate use.

buprenorphine use.

**4.3 Quality assurance of the analysis**

45-minute delay in equilibration of morphine concentrations in saliva compared to plasma following intramuscular administration of 10- and 20-mg doses; thereafter, saliva concentrations paralleled plasma concentrations. Morphine can be detected in oral fluid following intravenous administration, the smoking of heroin and poppy seed ingestion. Positive tests for morphine and codeine (with higher codeine

*CODEINE*: a naturally occurring phenanthrene alkaloid and opioid agonist. It appears to be most commonly taken orally. While it is not a metabolite of morphine, it is metabolised by oxidation to morphine and norcodeine and by conjugation. Kim et al. [52] demonstrated that following oral administration of 60 and 120 mg, codeine appeared in oral fluid within an hour and reached maximum concentration in approximately 1.6–1.7 hours. Concentrations in oral fluid correlated significantly with plasma concentration and were three to four times higher in oral fluid than plasma. Codeine could be detected in oral fluid for approximately 21 and 7 hours at cut-off concentrations of 2.5 and 40 ng/mL, respectively. Following intramuscular codeine of 60 and 120 mg, codeine appeared rapidly in oral fluid and reached maximal concentrations in 0.5–0.75 hours. A positive test result for codeine and

*METHADONE*: a synthetic opioid used widely as an analgesic as well as in maintenance therapy for persons with opioid dependency. Methadone undergoes extensive metabolism in the liver to form cyclic metabolites, 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrrolidine (EMDP), and other minor metabolites. Methadone and EDDP appear rapidly in oral fluid and correlate with plasma concentrations. Therefore, the confirmed presence of oxidative metabolites such as EDDP and EMDP in oral fluid would be

*BUPRENORPHINE*: an orally available, semisynthetic opioid analgesic, used as a pain reliever and in the management of opioid dependence. Following sublingual administration, buprenorphine reaches maximal plasma concentrations in 1.3–1.6 hours. Its main metabolite is norbuprenorphine. Cone [48] reported measurements of buprenorphine in saliva following intramuscular and sublingual administration of single doses of buprenorphine. Drug concentrations in saliva were substantially lower than plasma at all times following intramuscular administration and were substantially higher following sublingual administration. The low S/P ratio following intramuscular administration is probably due to the high fraction of drug that is protein-bound in plasma. Close correspondence between saliva and plasma buprenorphine concentrations was observed in subjects who administered buprenorphine sublingually on a daily or every-other-day basis. If the oral fluid test reveals buprenorphine ≤ norbuprenorphine, this suggests chronic

Drug testing laboratories must implement a quality management system that includes all aspects of the testing process, such as sample reception, chain of custody, safety and reporting of results, screening and confirmation tests, certification

Hence, the laboratory should remain constantly updated on the evolution of analytical techniques, whether for finding new drugs or responding to requests for investigations concerning narcotic and pharmacological drugs. A highly qualified analytical chemical-toxicological laboratory depends on ISO/IEC 17025 [53] accreditation standards and procedures to demonstrate that it operates competently and generates valid results. These standards are agreed by experts the world over,

of calibrators and controls, and validation of analytical procedures.

**148**

thus promoting confidence in the work of accredited laboratories and other bodies, on national and international levels, and facilitating cooperation between them. With ISO certification, results are more widely accepted between countries without the need for further testing, consequently improving international trade.

Of course, the benefits of advanced equipment in the chemical-toxicological laboratory go hand in hand with the expertise of qualified personnel with specific competence and adequate scientific training (not restricted to the analytical chemical field). Only through this combination (state-of-the-art tools plus qualified personnel) will it be possible to develop new analytical methodologies in the field of toxicological-forensic analysis and respond to administrative, criminal and social needs imposed by the legal system.

#### *4.3.1 Validation of an analytical method for the detection of drugs in saliva*

The validation of analytical methods includes procedures designed to establish that a particular method, used for the identification and/or quantification of an analyte in a given biological matrix, is reliable and reproducible. It is a question of demonstrating that the performance characteristics of the method meet all the requirements for its intended purpose and application. Any analysis methodology used routinely by the laboratory must be previously validated according to internationally agreed procedures [17].

For the most commonly used screening tests, validation procedures are not usually necessary as the method is validated by the manufacturer. In any case, the analysis kit includes calibrators and controls which are to be inserted into each batch of samples to be analysed in order to verify the accuracy and precision of the analyses (according to predetermined target values). In the event that changes are introduced which deviate from the manufacturer's instructions (for example, the biological matrix used is not the one indicated by the manufacturer, variation of the quantification limit, etc.) the laboratory must carry out a complete validation of the method/modified kit. It is best to fully respect the instructions provided by the manufacturer in the use of a kit, or in any case modifications should only be carried out in cases where it is not possible to use other methods.

The analysis methodology can be used routinely by the laboratory if the calculated validation parameters fall within the limits established by the relevant international directives [54].

The use of a good internal quality programme guarantees the reliability of the analytical results and avoids any random errors that may occur in the analytical and/or pre- or post-analytical phase that may affect the accuracy of the result.

The laboratory must participate in appropriate external quality assessment programmes. Analytical performances outside the criteria established by the External Quality Assessment (EQA) programme must be promptly corrected. The choice of one programme over another must be made on the basis of the best scientific evidence obtainable. Participation may concern the identification of classes of substances or individual substances and quantification in the case of confirmatory analyses according to the legal cut-offs or established by the management body of the programme.

In the case of screening tests, the expression of the results is generally in terms of "positive" or "negative". In the case of confirmatory analysis, it is necessary to provide not only qualitative but also quantitative data, namely the concentration detected according to a given calibration curve for the analyte identified in the saliva sample. The results of participation in the EQA are useful for the laboratory director and staff in helping to gauge the performance of the laboratory. In the event of errors, it is important to identify the causes and implement corrective actions that prevent them from recurring.