in fact many analytical methods , employing different techniques , are being developed for evaluation of pharmaceutical preparations .
vitamins of b group were well separated by rp - hplc . a method for the simultaneous determination of taurine and 10 water - soluble vitamins including vitamin b1 ( thiamine ) , b2 ( riboflavine ) , b5 ( pantothenic acid ) , b6 ( pyridoxine and pyridoxal ) , b8 ( biotin ) , b9 ( folic acid ) , c ( ascorbic acid ) , and b3 ( nicotinamide and nicotinic acid ) in multivitamin tablets was developed and validated .
some water - soluble vitamins of our interest ( ascorbic acid , thiamine hydrochloride , riboflavine-5-phosphate sodium , pyridoxine hydrochloride , nicotinamide , and ( + ) -panthenol ) and two preservatives ( methylparaben and sodium benzoate ) in multivitamin syrup were well - separated on zorbax sb - aq ( c18 ) column .
a hplc - uv method was established for the simultaneous determination of eight vitamins , including b1 , b2 , b3 , b6 , b9 , cyanocobalamin , ascorbic acid in baby milk powder . a validated hplc - uv method for the determination of seven b - complex vitamins ( b1 , b2 , b3 , b6 , b9 , and cyanocobalamin ) in pharmaceuticals and biological fluids after solid - phase extraction was performed .
simultaneous determination of water- and fat - soluble vitamins in pharmaceutical preparations by hplc was performed in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of trifluoroacetic acid and methanol .
vitamins analyzed were b1 , b2 , b5 , b6 , b9 , b3 , and b12 ( cyanocobalamin ) .
the rp ion - pair hplc method was applied for determination of some vitamins of our interest in multivitamin with minerals from different authors . for nicotinamide , pyridoxine hydrochloride , thiamine mononitrate and riboflavine a hplc method
a sensitive rp - hplc method was developed and validated for the simultaneous determination of b1 , b3 , b6 , and b9 in pentovit coated tablets .
the procedures were carried out on a supelcosil abz column with methanol , heptanesulfonic acid sodium salt , and triethylamine as the mobile phase .
for the determination of thiamine and riboflavine in duoweiyuansu tablet an hplc - uv method was established . the mobile phase consisted of ion - pairing reagent ( containing 1-hexanesulfonate , glacial acetic acid , and triethylamine)/methanol ( 80  :  20 ) .
the method can be used for the quality control of thiamine and riboflavine in duoweiyuansu tablet .
there are many papers about vitamins of b group analysed by capillary electrophoresis ( ce ) and its modifications .
some authors studied determination of vitamins in food based on supercritical fluid extraction prior to micellar electrokinetic capillary chromatographic ( mekc ) analyses of individual components .
the method was optimized using sodium cholate as the micellar phase for the separation of 11 b - vitamins , ascorbic acid , and 4 impurities in about 25  min .
other authors investigated in capillary zone electrophoresis ( cze ) analysis the effect of lower organic alcohols as cosurfactants ( methanol , ethanol , n - propanol , isopropanol , propanediol , n - butanol and isoamyl alc . ) and n - hexane as an organic modifier in phosphate buffer with varying sds concentration using a set of vitamins and p - hydroxybenzoic acid as the test mixture .
the cze analyses of three vitamins ( b1 , b2 , and b6 in tablets achieved using phosphate - borate buffer ph 9.0 short time of analysis ( 5  min ) .
one year later the same authors studied another specific , precise , sensitive , and accurate method for separation of the same vitamins obtaining high separation efficiency and shorter analysis time ( 3  min ) .
four b vitamins , as b1 , b2 , pyridoxal , pyridoxine , and pyridoxamine ( b6 ) in a pharmaceutical product were determined simultaneously using cze .
an hcl soln . was used for the extraction of the vitamins from a multivitamin - multimineral tablet .
mixture of four water - soluble b - group vitamins were analysed both by cze and meck .
the quantitative analyses of different pharmaceutical formulations were compared with the lc method of the us pharmacopeia obtaining a good correlation .
rapid methods were developed for simultaneously separation of five vitamins : b1 , b2 , b6 , nicotinamide , nicotinic , and ascorbic acid .
they were tested on 15 real samples obtaining good resolution by ( cze ) and ( mecc ) .
cze was performed with 0.02  m borate buffer , while mecc in 0.02  m borate / phosphate buffer with 4% acetonitrile containing .
some authors studied a method for the analysis of six water - soluble vitamins ( thiamine , nicotinamide , riboflavin , pyridoxine , pantothenic , and ascorbic acid ) in a pharmaceutical formulation , by cze . a good compromise between resolution , analysis time , and analyte stability
this cze method was very useful for the separation of more complex samples , but cyanocobalamin could not be separated from nicotinamide in this cze system .
good results with respect to linearity , precision , and accuracy were obtained in the concentration range studied for the 6 vitamins .
spectrophotometric determination of ternary mixtures of thiamine , riboflavin , and pyridoxal in pharmaceutical and human plasma by least - squares support vector machines were performed .
the partial least squares ( pls ) modelling and least - squares support vector machines were used for the multivariate calibration of the spectrophotometric data .
the same procedure was applied successfully for determination of b1 , b2 , b3 , and b6 in pharmaceuticals by other authors .
simultaneous determination of fourteen water - soluble vitamins ( 13 of b - group and vitamin c ) in selected food matrices by lc / ms / ms technique is described in a recent paper .
analytes were separated in less than 10  min with recoveries between 30% and 70% .
the qc process in pharmaceutical industry needs methods that are able to determine , in a single run , the majority of components . in many cases , to determine all components of a multivitamin pharmaceutical preparation , it is necessary to perform different analyses applying different techniques .
capillary electrophoresis ( ce ) is a powerful analytical technique that is widely used in research , and in quality control of pharmaceuticals . as demonstrated exploring the literature data , the mekc was the only method   able to separate up to 11 b - group vitamins .
other ce methods offer the possibility to separate only a few ( max 5 ) molecules of this class .
there are no publications about simultaneous determination of nine b - group vitamins by cze , the simplest in ce system , which offers several advantages over hplc such as rapid analysis , lower solvent consumption , then lower costs and minor environmental impact , and higher efficiency . for this purpose ,
the aim of this work is to develop a validated cze method for assay of a multicomponent pharmaceutical and veterinary formulations in routine analysis .
the analyses were carried out on a p / ace system from beckman instrument fullerton , ca ( usa ) , with a uv - dad detector . for ruggedness evaluation , electrophoretic separations
were carried out on the spectraphoresis 1000 instrument from thermo - quest corporation , ca ( usa ) .
the uncoated fused silica capillary i d = 50  m , 59.5  cm total length and 49.5  cm effective length was supplied by sge ( melbourne , australia ) .
the sample injections were performed in a hydrodynamic mode ( 5  s under 0.5  psi ) . all reagents were of analytical grade purity .
borax , disodium hydrogen phosphate dihydrate , sodium dihydrogen phosphate monohydrate , potassium chloride , and ctab ( cetyl trimethylammonium bromide ) were from fluka ( buchs , switzerland ) .
phosphoric acid , sodium hydroxide , chloric acid , and acetonitrile were supplied by carlo erba ( milan , italy ) .
sodium bicarbonate , citric acid , oxalic acid , and sodium bitartrate monohydrate were from sigma - aldrich ( steinheim , germany ) .
the real samples were commercially available pharmaceutical products : berocca plus ( bayer s.p.a ) , gabbrovital b forte ( ceva vetem spa ) , trinidex ( ldb lab.diaco biomedicali spa ) , and biochetasi ( sigma - tau spa ) . for solution filtering the syringe filters 0.45  m (
thiamine hydrochloride ( b1 ) , thiamine pyrophosphate chloride ( b1a ) , riboflavine ( b2 ) , riboflavine-5monophosphate ( b2a ) , nicotinamide ( b3 ) , d - pantothenic acid calcium salt ( b5 ) , pyridoxine hydrochloride ( b6 ) , biotin ( b8 ) , folic acid ( b9 ) , b10 ( 4-aminobenzoic acid ) , cyanocobalamin ( b12 ) , hydroxocobalamin ( b12a ) , and 2 - 3 dihydroxybenzoic acid were purchased from fluka ( buchs , switzerland ) .
standard stock solutions of studied analytes ( at 1  mg / ml concentration ) were prepared in distilled water , riboflavine and folic acid were dissolved in hcl , respectively , 3  m and 2  m. the calibration solutions were obtained by dilution of the stock solution with distilled water to give a desired analyte concentration .
the quantity of powder equivalent at one tablet was weighted and dissolved in a 50  ml volumetric flask with distilled water .
after sonication for 10  min in an ultrasonic bath , the supernatant solution was centrifuged for 5  min and then filtered through the 0.45  m syringe filter .
finally the solution was diluted with distilled water to reach a concentration order inside the calibration range . for trinidex sample , the intravenous injection solution was filtered through the 0.45  m syringe filter without dilution and injected .
for gabbrovital preparation , the veterinary intramuscular injection solution was diluted 1  :  1000 because of its high concentration , then filtered and analysed . for biochetasi sample ,
the powder contained in the vial for the intramuscular injection , was diluted with distilled water to reach a desired concentration and then filtered through the 0.45  m syringe filter .
in order to propose a suitable method for routine analysis , it was necessary to evaluate the experimental conditions for the best resolution of studied analytes .
first , because of different solubility and stability for each vitamin , many conditions for their dissolution were tested .
several parameters for the cze analysis , such as bge composition and concentration , ph , injection conditions , wavelengths , applied voltage , and temperature were explored .
the bge buffer composition is one of the most important parameters in capillary electrophoresis . in order to obtain maximum precision level , it is necessary to choose the buffer very carefully .
most of vitamins are nitrogen - containing , protonable molecules , so starting point of this work was to explore the low range of ph values for their separation . in the preliminary step of study , the bges tested ph ranging between 1.54.2 and concentration ranging between 20  mm to 50  mm , are hcl / kcl , phosphate , phosphate - oxalate , phosphate with 5% acetonitrile , phosphate with ctab , phosphate - tartrate , and tartrate . only in 20  mm phosphate buffer at ph
= 2.3 good separation of eleven analytes was realised in about 23 minutes . in figure 1 , analytes from 1 to 7 are cations , the others ( 810 ) are in anionic form . in these operating conditions ,
riboflavine ( b2 ) and cyanocobalamin ( b12 ) are overlaid . since these compounds are often contained in the same pharmaceutical preparation , it is impossible to determinate both of them .
long analysis time , high intensity of current , and low repeatability of migration time ( rsd% ~ 10% ) did not allow to propose this procedure for routinely analyses .
bge buffers at ph between 8.5 and 9.3 into the range of concentration 20  mm to 80  mm were explored : tris / hcl , bicarbonate / carbonate , tris / borate , borate , borate with edta , and borate with ctab .
best separation of nine vitamins was realized in 20  mm borate buffer at ph 9.2 in about 9 minutes ( figure 2 ) .
the peaks are symmetric and baseline resolved for individual b vitamins with exception of d - pantothenic acid calcium salt ( b5 ) and riboflavine-5monophosphate ( b2a ) , that are coeluted .
since a good compromise between peak symmetry , resolution , and analysis time resulted in 20  mm borate buffer at ph = 9.2 , these experimental conditions were chosen as optimal .
primarily it was necessary to select the most suitable wavelength for the simultaneous detection of vitamins , since spectral properties of each one differs enough .
the uv spectra obtained by uv - dad show three suitable wavelengths for quantitative analysis .
214  nm for all the vitamins , at exception of d - pantothenic acid calcium salt ( b5 ) and riboflavine-5monophosphate ( b2a ) , that are overlaid .
then the quantitative analysis of these two analytes was realized at the wavelengths of 190 and 260  nm , because the considered compounds do not interfere themselves .
in fact vitamin b5 has only maximum of absorbance at 190  nm , while vitamin b2a presents two maximums at 214 and 260  nm .
for the optimization of separation voltage the analyses ranging the applied voltage between 10 and 30  kv were carried out . at low voltage
the separation of all the analytes was reached but the analysis time increased to 20  min with the broadening of peaks . increasing the voltage ( max 30  kv ) the optimal value results in 20  kv producing the current intensity of 25  ua and good repeatability . at 30 
the increase of temperature ( 30c ) produced the shortening of analysis time ( the viscosity of the bge decrease ) .
it should be advantageous setting high temperature of cartridge but b - complex is thermolabile .
the precision was evaluated in terms of rsd% of migration time ( t 
 m ) and corrected peak area ( a 
 c ) for intraday and interday analyses .
for the intra - day precision evaluation the standard solutions at three concentrations levels ( low , intermediate , and high ) were injected in the same day . to estimate the interday precision ,
the standard solutions were analysed for five consecutive days performing five consecutive injections every day .
the linearity of detector response was tested in different ranges as reported in table 2 . for the quantitative analysis
six standard solutions , containing nine analytes of interest and the internal standard , were injected in triplicate .
the calibration lines were obtained plotting r ( corrected areas ratio ) versus the standard solutions concentration using microsoft excel .
lod , the lowest concentration of analytes that can be distinguished from the noise , defined as signal to noise ratio
s / n of 3  :  1 was ranging between 0.9  g / ml to 9.0  g / ml .
loq , the lowest concentration of analytes that can be quantified with good precision , defined as signal to noise ratio s / n of 10  :  1 was ranging between 3.0  g / ml to 30.0  g / ml .
rsd% of migration time and corrected peak area values were lower , respectively , than 0.5% and 3.0% .
a typical electropherogram of commercial pharmaceutical preparation ( berocca plus ) is shown in figure 3 .
after fortification , the real sample undergo the analysis and the resulted concentration were compared with the data obtained without fortification .
for this purpose , measurements on the spectraphoresis 1000 apparatus in the optimal experimental conditions were performed . in this instrument
the cartridge form is different and the capillary total length is 44  cm , the effective length 36  cm ( shorter than optimal length of capillary utilized on the beckman instrument ) .
consequently , on this instrument , it was necessary to apply lower voltage ( 10  kv ) .
the electropherogram , obtained on this apparatus , injecting the standard mixture in hydrodynamic mode for 1  sec , is shown in figure 4 .
proposed method was developed for the simultaneous determination of nine water - soluble vitamins ( b1 , b1a , b2 , b2a , b3 , b5 , b6 , b9 , and b10 ) in the multivitamin pharmaceutical formulations by cze .
the best resolution was obtained in about 9  min using a simple tetraborate buffer 20  mm , ph = 9.2 at 25c , and constant voltage of 20  kv in uncoated fused silica capillary .
the method is suitable for the vitamins b analysis as the validation confirmed : it is precise , accurate , and rugged .
the analyses of real samples proved its applicability . since no expensive reagent and no pretreatment of samples
are required in this procedure , it offers a valid alternative for quality control analysis in the pharmaceutical industry .
 a competitive cze method for quality control analysis of multivitamin preparations and veterinary products containing b - group vitamins was developed . 
 vitamins of interest are thiamine hydrochloride ( b1 ) , thiamine monophosphate chloride ( b1a ) , riboflavine ( b2 ) , riboflavine-5monophosphate ( b2a ) , nicotinamide ( b3 ) , d - pantothenic acid calcium salt ( b5 ) , pyridoxine hydrochloride ( b6 ) , folic acid ( b9 ) , and 4-aminobenzoic acid ( b10 ) . 
 these analytes were separated optimizing the experimental conditions in 20  mm tetraborate buffer ph = 9.2 as a bge ( background electrolyte ) , on a beckman p / ace system mdq instrument , using uncoated fused silica capillary . 
 
 the effective capillary length was of 49.5  cm , i.d . 
 = 50  m , the applied voltage 20  kv and the temperature 25c . 
 detection was performed by a diode array detector at 214  nm for all vitamins except b5 ( 190  nm ) and b2a ( 260  nm ) . separation time was about 9  min . after experimental conditions optimization , the proposed method was validated . 
 precision of migration time and corrected peak area , linearity range , lod and loq , accuracy ( recovery ) , robustness , and ruggedness were evaluated for each analyte demonstrating the good reliability of the method . 
 analyses of the pharmaceutical real samples were performed and confirmed the versatility of this method . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion