analysis of biotin

Journal of Chromatography A, 881 (2000) 331343www.elsevier.com/ locate /chroma

Review

Analytical techniques for determining biotina a,b a aEvangelia Livaniou , Danae Costopoulou , Irene Vassiliadou , Leondios Leondiadis ,

a ,1 a,b , a*John O. Nyalala , Dionyssis S. Ithakissios , Gregory P. EvangelatosaImmunopeptide Chemistry Laboratory, Institute of Radioisotopes /Radiodiagnostic Products, National Center for Scientific Research

Demokritos, Athens 153 10, GreecebPharmaceutical Technology Laboratory, Department of Pharmacy, University of Patras, Patras 261 10, Greece

Abstract

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years,the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as theinherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosisof biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial.Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplementproducts, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in varioussamples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In thisreview, the most representative of these methods are presented, and their characteristics are evaluated. 2000 ElsevierScience B.V. All rights reserved.

Keywords: Reviews; Food analysis; Pharmaceutical analysis; Biotin; Vitamins

Contents

1. Introduction ............................................................................................................................................................................ 3322. Analytical techniques for biotin ................................................................................................................................................ 334

2.1. Microbiological methods ................................................................................................................................................. 3342.2. Bioassays ....................................................................................................................................................................... 3342.3. Physicochemical methods ................................................................................................................................................ 3342.4. Binding assays................................................................................................................................................................ 3372.5. Other suggested analytical methods for biotin ................................................................................................................... 338

3. Discussion .............................................................................................................................................................................. 3384. Nomenclature ......................................................................................................................................................................... 341Acknowledgements ...................................................................................................................................................................... 341References .................................................................................................................................................................................. 342

*Corresponding author. Tel.: 130-1-6513021; fax: 130-1-6510594.E-mail address: [email protected] (D.S. Ithakissios)1Current address: Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

0021-9673/00/$ see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0021-9673( 00 )00118-7

332 E. Livaniou et al. / J. Chromatogr. A 881 (2000) 331 343

1. Introduction

Biotin (hexahydro-2-oxo-1 H-thieno[3,4-d]imid-azole-4-pentanoic acid) is a water-soluble vitaminbelonging to the B-complex, which is found in smallquantities in all living cells. It exists in eight isomerforms, but only D-(1)-biotin is biologically active.

Biotin is synthesized in various bacteria andhigher plants [1,2]. However, several microorga-nisms as well as higher animals are not capable ofsynthesizing biotin and their needs in this vitamin aremet by dietary intake [3]. In addition, biotin can bebiosynthesized in the intestinal tract of higher ani-mals by symbiotic microorganisms [3,4]. In theintestine, biotin is absorbed through a saturabletransportation system, while at great biotin concen-trations, passive diffusion predominates. Exit ofbiotin from the enterocyte is carrier-mediated. Assome studies suggest, the enzyme biotinidase, whichis involved in the in vivo recycling of the vitamin,serves as a carrier protein for the transport of biotininto the cell as well as a biotin binding protein inserum [5].

In nature, biotin is found either free or covalentlybound to proteins or peptides. The catabolism of freebiotin in microorganism cells has been studiedextensively. On the other side, little is knownconcerning catabolism of free biotin in mammaliancells. It is generally believed that biotin is catabol-ized by b-oxidation at its side chain. This leads tothe formation of bisnorbiotin, tetranorbiotin andrelated metabolites. The sulfur in the heterocyclicring can become oxidized to biotin-L-sulfoxide, Fig. 1. Structural formulae of biotin and its main metabolites.biotin-D-sulfoxide and biotin sulfone. In mammals,degradation of the heterocyclic ring, leading mainlyto dethiobiotin, is quantitatively minor. The structur-al formulae of biotin and main biotin metabolites are and decarboxylases, which catalyze the incorpora-shown in Fig. 1. These metabolites, together with tion, transfer or elimination, respectively, of a car-non-identified biotin derivatives and biotin, are urine boxyl group to (or, from) a biomolecule. Theseexcreted. Regarding the metabolism of biotin co- biotinyl enzymes catalyze significant metabolic re-valently bound to peptides and proteins, it is believed actions and are involved in important biochemicalthat they are initially hydrolyzed to small biotinyl processes, such as gluconeogenesis, lipogenesis,peptides, which consequently release biotin through aminoacid metabolism and energy transduction [5].biotinidase action [5]. In animal cells, the following biotinyl carboxylases

Intracellularly, the most important biochemical have been found: acetyl-coenzyme A(CoA) carboxy-function of biotin is that of the coenzyme of certain lase (E.C. 6.4.1.2), pyruvate carboxylase (E.C.enzymes, known as carboxylases, transcarboxylases 6.4.1.1), propionyl-CoA carboxylase (E.C. 6.4.1.3),

E. Livaniou et al. / J. Chromatogr. A 881 (2000) 331 343 333

and b-methylcrotonyl-CoA carboxylase (E.C. volvement in severe animal syndromes, such as the6.4.1.4). In biotinyl enzymes, biotin is covalently avian Fatty Liver and Kidney Syndrome (FLKS) andattached via its carboxyl group to a lysyl -amino the trout blue slime disease [3,17,19,20].group of the corresponding apoenzyme, the reaction Prolonged biotin deprivation in humans leads tobeing catalyzed by certain enzymes reported as pathological symptoms, which are eliminated whenbiotin holocarboxylase synthetases [6]. Biotin is biotin is administered [3,21].liberated from biotinyl enzymes through the catalytic Biotin deficiency has been related to seriousaction of the enzyme biotinidase [7]. Biotin de- human disease states, such as the inherited metabolicficiency can lead to decreased catalytic activity of syndrome Multiple Carboxylase Deficiency (MCD).biotinyl enzymes, which may be manifested by This syndrome, which is accompanied by seriousclinical symptoms, mainly neurological and dermato- clinical symptoms, can be caused by defectivelogical ones [3]. Particularly severe are the neuro- activity of the enzymes involved in the in vivo biotinlogical symptoms, which, along with other findings, recycling [22,23]. Other human malfunctions, e.g.may indicate that biotin is especially important for Sudden Infant Death Syndrome (SIDS), seborrheicthe correct functioning of nervous tissue [8,9]. Biotin dermatitis of infancy, Leiner disease and Retthas been demonstrated to increase guanylate cyclase syndrome have been associated with biotin de-activity in primary cultures of rat hepatocytes ficiency status [3,24].[10,11], leading to an increase in hepatic glucokinase Administration of biotin in pharmacological dosesactivity and mRNA levels [12,13]; it has also been leads to elimination or at least alleviation of clinicalshown to affect glucokinase activity and mRNA symptoms in neonates with MCD [23]. Moreover, inlevels in cultured pancreatic beta cells, which may cases of patients with non-insulin dependent diabetesindicate the vitamins direct influence on glucose mellitus, administration of biotin has been reportedutilisation [14]. Moreover, biotin has been reported to lead to significant decrease in blood glucose levelsto enter the human cell nucleus, where it may be [25]. Biotin treatment has been reported to improveinvolved in histone biotinylation [15], although this metabolic malfunctions occuring in patients withfunction has not yet been completely elucidated. sternocostoclavicular hyperostosis [26]. Biotin hasRecent findings indicate that biotin levels and ex- also been reported to alleviate clinical symptoms ofpression of related genes are decreased in cancer patients under long-term hemodialysis treatmenttissues [16]. [27]. As reported, biotin has been used successfully

The richest dietary sources of biotin include liver, for the treatment of acne, seborrheic eczema andkidneys, heart, pancreas, poultry, egg yolk and milk. alopecia [3]. So far there has been no indication ofSmaller amounts are found in plants, mainly in seeds adverse effects due to high doses of biotin adminis-[17]. The dietary biotin intake in humans living in tration in humans.western countries has been estimated to be 35 to 70 Decreased biotin status, as compared to that of

21mg day , which seems to be absorbed almost normal population, has been reported to occur incompletely [5]. A dietary intake of 35 mg of biotin several population groups, such as pregnant womenper day is considered adequate for healthy adults. [28,29]. An active transport mechanism of maternalHowever, certain population groups, such as preg- biotin seems to exist in favor of the fetus [30].nant or lactating women, patients under anticonvul- Neonate serum biotin levels were found to depend onsant therapy, etc., may need increased biotin intake the kind of milk they were fed with [31]. The content[5]. Vegetarian diet does not seem to affect biotin of human milk in biotin and biotin metabolites hasstatus [18]. been extensively studied [3234]. Decreased circul-

Low biotin intake has been reported to result in ating biotin levels have been also reported in pa-serious biochemical disorders in animal organisms, tients on total parenteral nutrition [3537], patientssuch as reduced carboxylase activity, inhibition of receiving certain anticonvulsants [38], gastrectomiz-protein and RNA synthesis, reduced antibody pro- ed patients, alcoholics and children with severe burnsduction, etc. There are indications of biotin in- [3]. The exact clinical consequences or practical


utility of this finding are not, however, completely ated by Kloeckera brevis cells, in the presence ofclarified. varying quantities of biotin [43]. Microbiological

It is evident that diagnosis of biotin deficiency as methods have been applied to determining biotin inwell as monitoring of biotin levels of patients various materials, including food products, seawaterreceiving biotin treatment is very crucial. Equally and biological fluids, such as serum, urine and tissueimportant is the determination of biotin levels in extracts [40,4446].food and food supplement products. For this reason,analytical methods have been developed, in order to 2.2. Bioassaysdetermine biotin in biological fluids, as well asvarious kinds of food products and pharmaceutical These assays, which are usually less sensitive thanpreparations containing biotin. the microbiological ones, were mainly applied to the

determination of biotin in food material, more spe-cifically the bioavailable fraction. In biotin bioassays,

2. Analytical techniques for biotin rats or chickens, whose development has been in-hibited by artificial biotin deprivation, are fed with

The analytical techniques reported so far in the pure biotin, as reference, or with samples of un-literature for determining biotin can be divided into known vitamin content [3,39]. The determination ofthe following main categories: microbiological meth- the concentration of the unknown sample is based onods, biological methods (bioassays), physicochemical the animal development curve, which expressesmethods and binding assays. weight gain in relation to the logarithm of the

quantity of biotin administered.2.1. Microbiological methods In biotin bioassays one could also include methods

that determine biotin indirectly through its biological29 21These highly sensitive methods (10 g l ) are function, i.e. by assessing the activity of a special

among the first assays developed for determining biotin dependent enzyme, e.g. pyruvate carbox-biotin. They are based on the principle that growth of ylase or biotinyl CoA synthetase, in the presencemany microorganisms depends upon the presence of of increasing concentrations of biotin [3,4749].biotin in the culture media, since this vitamin is anessential nutrient for the microorganism under culti- 2.3. Physicochemical methodsvation. Thus, the microbiological determination ofbiotin is achieved by inocculating a certain micro- Physicochemical methods for determining biotin,organism culture with standard solutions or unknown due to their rather limited sensitivity, are mainlysamples of the vitamin, and then quantifying the applied to the measurement of the vitamin in samplesculture development, e.g. by turbidimetry or titration with high biotin concentrations, such as pharma-[3,39]. Many microorganisms have been applied to ceutical multi-vitamin preparations. These includethe development of microbiological assays for biotin, methods of spectrophotometry, colorimetry, polarog-e.g. Lactobacillus plantarum, Lactobacillus casei, raphy, thin layer chromatography, gas chromatog-Saccharomyces cerevisiae, Ochromonas danica, Mi- raphy, liquid chromatography, capillary zone electro-crococcus sodonensis, Neurospora crassa, phoresis (Table 1) and high-performance liquidRhizobium trifolii, Escherichia coli C162, etc. chromatography (Table 2).[40,41]. Microbiological methods have also been Among the first physicochemical methods de-developed based on the combined use of micro- veloped to determine biotin was the spectrophoto-organisms and radiolabelled compounds. For exam- metric one developed by Green in the mid 60s [50].

14ple, the incorporation of CO in microorganism This method is based on the red shift (from 280 nm2cells is determined in the presence of varying to 233 nm) observed on the absorption spectrum ofquantities of biotin [42]. In addition, a method has the tryptophan residues of avidin, a glycoprotein that

14been reported which determines the amount of CO specifically and stoichiometrically binds biotin with a214 215 21(metabolic product of L-[1- C]-methionine) gener- K of 10 mol l [51], upon binding of biotin. Aaff


Table 1Physicochemical methods for biotin determination: Non-HPLC methods

Type of method Method description Original Ref.application(s)

Spectrophotometric Measurement of absorbance at 233 nm of [50]tryptophane residues of avidin in avidinbiotin complex

Spectrophotometric Measurement of decrease in absorbance at 500 nm [50](Colorimetric) after dissociation of avidinHABA complexSpectrophotometric Measurement of absorbance at 533 nm of colored product(Colorimetric) of reaction of biotin with p-dimethylaminocinnamaldehyde [52]Spectrophotometric Measurement of absorbance at 520 nm of iodine produced(Colorimetric) after oxidation of the S atom of biotin with potassium iodate [53]Spectrophotometric Measurement of absorbance at 350352 nm of tri-iodide [54]

produced after reaction of biotin with periodatePolarographic DMFwater and 0.05 M KNO mixtures of biotin [55]3

were used for polarographic determinationPaper Biotin, was identified using various solvent [56]chromatography and visualizing spraying systemsGas chromatography Biotin, was transformed to a silyl ester derivative Agricultural premixes and [57,58]

and underwent gas chromatography pharmaceutical preparationsThin-layer Biotin was visualized by spraying with Multi-vitamin preparations [59]chromatography p-dimethylaminocinnamaldehydeLiquid A Vydak HS C column was used for the separation of Multi-vitamin preparations [60]18chromatography biotin which was detected at low wavelength containing at least 300 mg of biotin eachLiquid chromatography Biotin and dethiobiotin were derivitized as methyl esters, Extracts of [61]Mass spectrometry separated on a HP RP 8 column and detected by MS. Bacillus sphaericus

Detection limit was lower than 10 ng.Capillary zone Solid biotin preparations were dissolved in double-distilled Pharmaceutical formulations [62]electrophoresis water and filtered before analysis.

colorimetric-spectrophotometric assay for determin- with periodate, consequent reaction of the iodateing biotin was developed by Green, also [50]. This formed with iodide, and photometric determinationassay is based on the removal of the dye p-hydroxy- of the tri-iodide finally produced [54].azobenzene-2-carboxylic acid (HABA) from the Polarography in DMFwater and 0.05 M KNO3avidin-HABA complex and the subsequent decrease mixtures was also used to measure biotin down toin the corresponding absorbance values at 500 nm, in the microgram level [55].the presence of increasing amounts of biotin. Among Paper chromatographic data using various solventthe first colorimetric assays developed for biotin was systems were also reported for biotin [56].also that of McCormick and Roth [52]. This is based Determination of biotin by gas chromatography,on the reaction of biotin with p-dimethylaminocin- after formation of the biotin silyl-esters, was reportednamaldehyde and the subsequent photometric de- in the literature in the early 1970s [57,58]. Gastermination of the colored product at 533 nm. chromatography was applied to determining biotin inAnother colorimetric assay for biotin was that in- agricultural premixes and pharmaceutical injectablevolving the oxidation of the sulfur atom in the biotin preparations [57].molecule to the corresponding sulphone with potas- A thin-layer chromatography method was alsosium iodate and the concurrent reduction of iodate to described for determining biotin [59]. This methodiodine, which is then extracted in cyclohexane and uses spraying with p-dimethylaminocinnamaldehydeits absorbance measured at 520 nm [53]. A later for visualizing biotin and was applied to the de-spectrophotometric assay for biotin measurement, termination of biotin in multi-vitamin preparations.characterized by enhanced sensitivity down to the A liquid chromatography method, based on themicrogram level, is based on the reaction of biotin use of a Vydak HS C column, was reported [60]. In18


Table 2Physicochemical methods for biotin determination (continued): HPLC methodsDetection system Type of column Derivatization Detection limit Original Ref.

application(s)UV or

28Fluorescence mBondapak C BAP or BAP: 10 g Pharmaceutical products [63]1829Mmc esters Mmc: 10 g

Fluorescence Nucleosil C 9-Anthryl-diazomethane 1 ng [64]1826UV mBondapak C 10 g [65,66]18

Aquapore AX-300/Partisil SAX

Electrochemical LiChrosorb RP 18 5 10 ng Multivitamin preparations [67]detection HiberUV Zorbax ODS Multivitamin preparations [68]Fluoresence LiChrosorb Si 60 1-Pyrenyl-diazomethane Normal-phase: Pharmaceutical preparations, [69]

21100 fmol injection Serum spiked with biotinTSK-gel 80 TM Reversed-phase:

2110 fmol injectionUV Supelcosil LC-8-DB Almond cultivars [70]UV TSK gel ODS Thiamine esters 10 ng Pharmaceutical preparations [71]

80 TMFluorescence Shandon Hypersil Panacyl ester Normal-phase: Rat tissues [72]

Hypersil ODS 10 pmolReversed-phase:100 pmol

28Fluorescence Microsorb C Post column: 1.8?10 M Multivitamin preparations [73,74]18Avidin FITC Horse-feed supplement

Cell culture extractInfant formula

27Fluorescence Microsorb C Post column: 5.0?10 M Commercial vitamin tablets [74]18Avidin ANS Horse-feed supplement

this method, biotin was detected spectrophotomet- either pre- or post- column, in an attempt to decreaserically at low UV wavelength. The method was assay detection limit. HPLC methods could clearlyapplied to the measurement of biotin in multivitamin discriminate between biotin and its main analogues,tablets containing at least 300 mg of biotin each. A e.g. dethiobiotin, biotin sulfoxides and biotin sulfone.liquid chromatography mass spectrometry method The above methods have been applied mainly to thefor determining biotin was also described, measuring determination of biotin in pharmaceutical mul-as low as 10 ng of the vitamin [61]. The method was tivitamin preparations. However, some of them haveapplied to measuring biotin in extracts of Bacillus been used for analyzing more complicated samples,sphaericus cultures. e.g. food material. In some reports sample pretreat-

A capillary zone electrophoresis method has been ment was necessary, depending on the sample ana-described in the literature [62]. This method was lyzed and the assay protocol developed.applied to the analysis of biotin in pharmaceutical Among the HPLC methods developed for biotin informulations. early 1980s was that of Desbene et al. [63], which

Various high-performance liquid chromatography used a reversed-phase mBondapak C column.18(HPLC) methods for determining biotin have been Biotin was detected after pre-column derivatiza-described so far, some of which are presented in tion with either UV absorbing (v-4-di-Table 2. Various columns and elution protocols have bromoacetophenone) or fluorescent (4-bromomethyl-been used in these methods. Biotin detection was methoxy-coumarin) reagents. Detection limit was

28 21 29 21achieved without any previous derivatization (UV) 10 g ml and 10 g ml , respectively. Theor after proper derivatization (UV, fluorescence), method developed could discriminate between biotin


and its main analogues, e.g. dethiobiotin, biotin reaction with thiamine to a fluorescent thiochrome.21

sulfoxides and biotin sulfone. In another method The detection limit was 10 ng injection . The[64], biotin was reacted with 9-anthryldiazomethane method could be applied directly to pharmaceuticaland the fluorescent derivative was determined by preparations without any sample pretreatment. In anHPLC using a reverse phase Nucleosil C column. HPLC method reported in the early 1990s, in which18The lowest biotin amount to be detected was esti- either normal or reverse phase column was used,mated to 1 ng. The method could be applied to the biotin was detected after pre-column derivatizationanalysis of pharmaceutical products. Underivatized with panacyl bromide [( p-(9-anthroyloxy)phenacylbiotin, biotin synthetic analogs and intermediates of bromide], in the presence of crown ether catalyst, asthe vitamins commercial synthesis were analysed by a fluorescent label [72]. Lowest amounts of biotinreversed-phase and anion-exchange HPLC in a meth- detected by normal (Shandon Hypersil column) andod later reported [65,66]. Separation of complex reverse phase (Hypersil ODS) HPLC was about 10biotin mixtures was best accomplished by RP-HPLC pmol and 100 pmol, respectively. The method was(mBondapak C column). Anion-exchange HPLC applied to the determination of biotin in biological18(Aquapore AX-300 and Partisil SAX columns) was samples, i.e. rat intestinal tissues. Prior to HPLC,successful in separating simple mixtures in a rela- samples were extracted with chloroacetic acid andtively short time and also in separating sulfoxide purified by a combination of solid-phase extractionisomers. The detection limit of the RP-HPLC sepa- on C cartridges, ion-exchange chromatography and18

26 21ration was 10 g ml . Underivatized biotin was thin-layer chromatography. More complicated sys-analyzed by reversed-phase HPLC (LiChrosorb RP tems for analyzing biotin, which combine HPLC18 Hiber column), using electrochemical, instead of separation and post column detection based on avidinUV, detection [67]. The lowest biotin amount to be binding, have also been described [73,74]. Briefly, indetected was estimated to be 510 ng. The method a reversed-phase HPLC method of this type, thewas applied to the determination of biotin in mul- biotin containing HPLC effluent was reacted withtivitamin pharmaceutical preparations. Underivatized avidin labelled with fluorescein isothiocyanatebiotin was also analyzed with reversed-phase HPLC (FITC), which leads to an enhancement of the(Zorbax ODS column), using low UV (203 nm) fluorescence intensity at 520 nm. The assay detection

28detection [68]. The method was applied to the limit was 1.8?10 M. The system was applied to thedetermination of biotin in multivitamin-multimineral determination of biotin in liquid vitamin preparationstablets. The samples were pre-cleaned on a Sep-Pak and horse-feed supplement. In another approach, theFlorisil column. In another method [69], biotin was biotin containing HPLC effluent competes with thereacted with 1-pyrenyldiazomethane (no catalyst was fluorescent probe 2-anilinonaphthalene-6-sulfonicnecessary) and the fluorescent derivative was ana- acid (2,6-ANS) for the avidin binding sites. Onlyzed with normal (LiChrosorb Si 60) and reverse release from avidin, the fluorescence emission ofphase (TSK-gel 80 TM) HPLC. The detection limit 2,6-ANS is shifted toward the red region, yielding a

21 21was 100 fmol injection or 10 fmol injection , decrease in fluorescence intensity at 438 nm, whichrespectively. The method was applied to the de- is proportional to the analyte concentration. The

27termination of biotin in multivitamin tablets as well detection limit was 5.0?10 M. The above systemas in serum spiked with biotin. An HPLC method, was applied to the determination of biotin in liquidusing a Supelcosil LC-8-DB column and UV de- vitamin preparations, vitamin tablets, horse-feedtection, was applied to the determination of un- supplement and a liquid infant formula.derivatized biotin in Italian almond cultivars [70].Prior to HPLC analysis, samples were de-fatted with 2.4. Binding assaysn-hexane extraction and pretreated on a cation-ex-change extraction column. In another HPLC method, During the last 25 years, many attempts have beenusing TSK gel ODS 80 TM column, biotin was made to apply binding assays to the determination ofdetected after post-column derivatization and fluores- biotin. Biotin binding assays are based on the samecence measurement [71]. Derivatization consisted of principle as immunoassays, which are widely used inchlorination of the biotin amide bond and consequent clinical laboratory practice, differing from them only


in the fact that they utilize a specific binding protein uses a suitable metastable molecular complex con-instead of an antibody. Binding assays, like immuno- sisting of HABA bound to a membrane and catalaseassays, have many advantages such as high sensitivi- labelled avidin. The metastable complex dissociatesty, and rapid and convenient performance. upon exposure to biotin in solution, which can be

29 27 21In all biotin binding assays reported so far, the thus quantitated (10 10 g ml ) [111].specific binding protein used is the glycoprotein Another biotin assay system was developed based onavidin or the structurally similar protein streptavidin the competition between biotin labelled with the[51]. As already mentioned, both avidin and strep- electrically active compound daunomycin and biotintavidin exhibit an extremely high K along with in standard solutions for avidin binding sites [112].affhigh specificity in their binding to the biotin mole- The assay developed is homogeneous, sincecule. Due to their exceptional characteristics, the daunomycin labelled biotin renders electrically inac-avidin biotin (or, streptavidin biotin) system has tive upon binding to avidin, as can be seen by thebeen used in a lot of applications in the fields of corresponding electrode response. Daunomycin wasbiochemistry, biotechnology, immunochemistry, im- substituted by another, less toxic, electroactive com-munology, molecular biology, etc [75,76]. pound, namely Nile Blue, in another electroactive

In addition to avidin or streptavidin binding binding assay system developed [113]. Biotin wasprotein, many other reagents, e.g. various labels and also measured through direct electrical detection inseparation means, have been used in the biotin another binding assay system [114]. According to itsbinding assays developed so far. Thus, radioactive principle, the uptake of conjugated to HRP biotinisotopes, enzymes, chemiluminescent or fluorescent (B-HRP) by an avidin containing, electron conduct-substances or latex agglutination particles have been ing redox hydrogel film on the surface of an elec-used as assay labels. Many of the assays developed trode rotating in a H O solution and poised at 10.12 2are heterogeneous solid-phase systems. However, V (Ag/Cl), produces a substantial electrocatalyticseveral homogeneous systems, mainly based on current. The current, resulting from H O elec-2 2enzyme, fluorescence and bioluminescence labels, troreduction to water, signals promptly the occur-have also been described. Some of the most repre- rence of the reaction between avidin and B-HRP.sentative biotin binding assays are presented in Dissolved biotin can be measured through its block-Tables 3 and 4. ing of the binding of B-HRP to the avidin redox

Many biotin metabolites bind to avidin and strep- hydrogel-coated electrode. Similar systems have alsotavidin, though typically with lesser affinity. Thus, been reported [115]. Recently, an electrically wiredalthough binding assays can determine biotin very amperometric immunosensor assay was developedspecifically, e.g. among many other vitamins, they for the determination of biotin [116]. The methodcannot totally discriminate between biotin and was based on the use of a redox polymerbiotin metabolites; such discrimination may, how- h[Os(bpy) (PVP) Cl]Cl, where bpy5bipyridyl,2 10ever, be desirable, especially if biological fluids are PVP5poly-4-vinylpyridinej co-immobilized with ananalyzed, e.g. during pharmacokinetic studies, etc. anti-biotin antibody on an electrode surface. TheThus, analytical systems which combine a binding polymer transfers electrons between the electrodeassay with a HPLC methodology were described by surface and biotin bound to the anti-biotin antibodyMock [77]. These analyses were applied to the on the sensing surface. The current produced ismeasurement of biotin in human urine, serum and expressed vs. biotin concentration (detection limit:

27milk [34,78,79] 6?10 M).

2.5. Other suggested analytical methods for biotin3. Discussion

More sophisticated assay systems, which use thebinding protein avidin or streptavidin, have been Microbiological assays were the first assay meth-developed during the last 15 years. For instance, a ods developed for determining biotin. These assaysbioaffinity sensor method was described. The method are very sensitive. However, they are not always


Table 3Radioisotopic binding assays for determining biotin

Detection signal Tracer Assay type Reference14

b-Radiation [ C]Biotin Heterogeneous [80]liquid-phase(bentonite suspension)

14b-Radiation [ C]Biotin Heterogeneous [81]

liquid-phase(ZnSO solution)4

14b-Radiation [ C]Biotin Heterogeneous [82]

solid-phase(avidin-cellulose disks)

3b-Radiation [ H]Biotin Heterogenous [83]

liquid-phase(dextran-coated charcoal)

3b-Radiation [ H]Biotin Heterogeneous [84]

solid-phase(nitrocellulose filters)


liquid-phase(bentonite suspension)


solid-phase(anti-goat IgG linked to agarose)

125g-Radiation [ I]Biotinyl-derivative Heterogenous [87]

liquid-phase(dextran-coated charcoal)

125g-Radiation [ I]Biotinyl-derivative Heterogeneous [88]

solid-phase(avidin-cellulose)

125g-Radiation [ I]Biotinyl-derivative Heterogeneous i.[28]

liquid-phase ii. [89,90][(i)avidin /anti-avidin antibody,(ii)PEG precipitation]

125g-Radiation [ I]Biotinyl-derivative Heterogeneous [91]

solid-phase(streptavidin-Sepharose 4B)

125g-Radiation [ I]Avidin Heterogenous [92]

solid phase (biotinylatedalbumin-coated microplates)

highly specific, since other nutrients, e.g. fatty acids, mainly applied to the estimation of bioavailablemay promote certain microorganisms growth, thus biotin present in food samples [3].interfering with assay results [40]. In addition, Physicochemical assays for determining biotinmicrobiological assays are rather tedious and time- were first developed in the mid 60s. Their sensitivi-consuming, which renders them not ideal for routine ty varies from rather limited to rather high, moreanalysis of great numbers of samples. Nevertheless, specifically from the mg level of the first developedmicrobiological assays were applied to the determi- spectrophotometric assays to the pg level of thenation of biotin in various samples, such as food recently developed HPLC methods using fluoromet-material, human biological fluids, tissue extracts, etc. ric detection. These assays are considerably simpler[40,46]. and faster than the microbiological ones, but not as

A few bioassay systems have been proposed in the simple and fast as the binding assays, which areliterature for determining biotin. These assays were therefore considered advantageous for the routine


Table 4Non radioisotopic binding assays for determining biotin

Detection signal Tracer Assay type Ref.

Enzyme activity Biotin-lysozyme Homogeneous [93](potentiometric measurement)Enzyme activity Biotin-G6 PDH Homogeneous [94](spectrophotometric measurement)Enzyme activity Biotin-AP Heterogeneous [95](spectrophotometric measurement) solid-phase

(biotinylated BSA coated microplates)Enzyme activity Biotin-pyrophosphatase Heterogeneous [96](spectrophotometric measurement) solid-phase

(biotinylated IgG coated microplates)Enzyme activity Biotin-HRP Heterogeneous [97](spectrophotometric measurement) solid-phase

(streptavidin coated microplates)Enzyme activity Biotin and HRP immobilized Heterogeneous [98](spectrophotometric measurement) on unilamelar vesicles solid-phase

(anti-biotin antibody coated plates)Enzyme activity Streptavidin-HRP Heterogeneous [99](spectrophotometric measurement) solid-phase

(biotinylated IgG coated microplates)Enzyme activity Streptavidin-HRP Heterogeneous [100](spectrophotometric measurement) solid-phase

(biotinylated IgG coated microplates)Enzyme activity Streptavidin-HRP Heterogeneous [101](spectrophotometric measurement) solid-phase

(biotinylated BSA coated microplates)Latex agglutination Biotin-coated latex particles Homogeneous [102](turbidity measurement)Fluorescence Biotin-fluorescein Homogeneous [103]Fluorescence Avidin-ANS Homogeneous [104]Fluorescence Avidin-fluorescein Homogeneous [105]

aFluorescence Streptavidin-fluorescein Homogeneous [106]Chemiluminescence Biotin-isoluminol Homogeneous [107]Chemiluminescence Biotin-ABEI Homogeneous [108](energy transfer) Avidin-fluoresceinBiochemiluminescence Biotin-aequorin HomogeneousBiochemiluminescence Biotin-aequorin Heterogeneous [109]

solid-phase [110](avidin coated solid particles)

a This fluorescence binding assay has been applied to a flow injection analysis system.

analysis of great numbers of samples. However, the Biotin binding assays are very sensitive. In addi-main advantage of physicochemical assays over the tion, since they are usually characterized by a simpleother biotin analytical methods is their better dis- assay protocol and can be used for the analysis of acriminating capacity between biotin and biotin me- great number of samples in a short period of time,tabolites, e.g. biotin sulfoxides, etc. Several physico- they are considered ideal for every-day routinechemical methods have been applied to the measure- laboratory use. The first biotin binding assays de-ment of biotin in various samples, including food veloped were radioactive ones. These assays werematerial, biological fluids and, especially, pharma- applied to determining biotin in various samples,ceutical preparations, e.g. multivitamin tablets. such as food material, tissue extracts, and, especially,


human fluids, e.g. human serum, plasma, CSF and ture supernates, etc., might provide important in-urine, obtained from various population groups. Non- formation, of either theoretical or practical interest,radioactive biotin binding assays were later de- in a lot of different studies, e.g. nutritional, bio-veloped. These assays presented the advantage of not chemical, pharmacokinetic or pharmacological ones.using radioactive tracers, while they retained their Many analytical methods have been proposed in thehigh sensitivity characteristics. Few of these assays literature for measuring biotin in standard solutionswere, however, applied to the measurement of biotin and unknown samples. One should carefully investi-in unknown samples, especially complex biological gate certain parameters, e.g. expected biotin levels inones. Concerning specificity of biotin binding assays, the sample to be analyzed, presence and kind ofthis is very high as far as discrimination capacity biotin-resembling substances in it, special samplebetween biotin and other biological substances, pretreatment if necessary (e.g. extraction, ultrasoni-different from biotin metabolites, is concerned even cation, pre-cleaning through a Sep-Pak column,if these substances show structural similarities with preparation of derivatives to facilitate detection etc),the biotin molecule, e.g. urea and uric acid, etc. desirable number of samples analyzed per timeHowever, biotin binding assays cannot discriminate period, etc., before selecting the most appropriateequally well between biotin and biotin metabolites, assay methodology.as already mentioned. To overcome this problem,some researchers developed and used systems com-bining HPLC and binding assay methodologies.

Some more sophisticated analytical systems for 4. Nomenclaturedetermining biotin have been reported in the litera-ture. These systems usually use avidin or streptavidin ABEI aminobutylethylisoluminolamong their main reagents along with specially 2,6-ANS 2-anilinonapthalene-6-sulfonic aciddesigned complex detection system. Functioning of AP alkaline phosphatasethese systems has not, however, been evaluated in B Biotinunknown samples. BAP Bromoacetophenone

Special sample treatment is sometimes necessary BSA Bovine serum albuminprior to the assay performance [117], depending on CSF Cerebrospinal Fluidthe nature of the sample, or the assay principle and DMF dimethyloformamideprotocol, or sometimes on both. For instance, pre- FITC fluorescein isothiocyanatetreatment is necessary for preparing various kinds of FLKS Fatty Liver and Kidney Syndromesamples to be analyzed by the Lactobacillus plan- G6 PDH 6-phosphoric glucose dehydrogenasetarum microbiological assay. Thus, pharmaceuticals HABA p-hydroxyazobenzene-2-carboxylic acidand premixes are homogenized and solubilized in HPLC high-performance liquid chromatographydiluted alkali, while food materials and other natural HRP horseradish peroxidaseproducts are heat extracted prior to the assay [117]. IgG immunoglobulin GPretreatment is also necessary before analyzing MCD Multiple Carboxylase Deficiencynatural samples, mainly food material, by various Mmc methylmethoxycoumarinphysicochemical methods as already described. MS mass spectrometrySerum sample pretreatment has also been included in SIDS Sudden Infant Death Syndromethe protocol of radioisotopic binding assays whichuse a radiolabelled biotinyl amide as the assay tracer,so as to inactivate any serum biotinidase activity thatmight lead to tracer cleavage and affect assay results Acknowledgements[87,28].

The measurement of biotin levels in various kinds The authors express their thankfulness to Professorof samples, among which food material, pharma- Hippocrates Yatzidis for his long existing excellentceutical preparations, human biological fluids, cul- collaboration in biotin research.


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analysis of biotin

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