Anal. Chem. 1995,67, 1014-1018

Class-Selective Detection System for Liquid Chromatography Based on the Streptavidin-Biotin Interact ion

Nathaniel G. Hentz and Leonidas G. Backs*

Department of Chemistty, University of Kentucky, Lexington, Kentucky 40506-0055

A protein binding assay (BA) was coupled with high- performance liquid chromatography (HPLC) to provide a highly sensitive postcolumn reaction detection system for the biologically important molecule biotin and its derivatives biocytin, biotin ethylenediamine, 6-(biotinoy- 1amino)caproic acid, and 6-(biotinoylamino)caproic acid hydrazide. This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay, a conju- gate of streptavidin with fluorescein isothiocyanate (strepta- vidin-FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin-FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The HPLC-BA system is more sensitive and selective than either the BA or HPLC alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respec- tively. Finally, the analysis of an infant formula and several cell culture media, with little or no sample preparation, demonstrated the analytical utility of this system.

Coupling high-performance liquid chromatography (HPLC) to other analytical techniques can provide additional levels of information about the separated analyte~.'-'~ For example, hyphenated techniques, such as HPLC-MS, HPLC-FTIR, and HPLC-NMR, not only allow detection of the analytes but also give structural information about the separated compounds. We

(1) Hirschfeld, T. Anal. Chem. 1980, 52, 297A-31% (2) Tomer, K. B.; Parker, C. E. J. Chromatogr. 1989, 492, 189-221. (3) Chau, Y. IC; Wong, P. T. S. Fresenius'Z. Anal. Chem. 1991, 339, 640-

645. (4) Huang, E. C.: Henion, J. D. Anal. Chem. 1991, 63, 732-739. (5) Smith, J. B.: Thevenon-Emeric, G.; Smith, D. L.; Green, B. Anal. Biochem.

(6) DiNunzio, J. E. J. Chromatogr. 1992, 626, 97-107. (7) Huddleston, M. J.; Bean, M. F.; Carr, S. A Anal. Chem. 1993, 65, 877-

884. (8) Mock, K; Hail, M.; Mylchrest, I.; Zhou, J.; Johnson, IC; Jardine. I. J.

Chromatogr. 1993, 646, 169-174. (9) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K; Wilson, I. D. Anal.

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1991, 193, 118-124.

(10) Takatera, K; Watanabe, T. Anal. Chem. 1993, 65, 3644-3646. (11) Tielrooy, J. A; Vleeschhouwer, P. H. M.; Kraak, J. C.; Maessen, F. J. M. J.

(12) Laborda, F.; De Loos-Vollebregt, M. T. C. ; De Galan, L. Spectrochim. Acta

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268.

1014 Analytical Chemisfiy, Vol. 67, No. 5, March 7, 7995

have recently demonstrated that the on-line coupling of a homogeneous binding assay @A) to HPLC can not only improve the detection limits but also provide a highly selective detection system for liquid ~hromatography.'~ This HPLC-BA system allows selective detection of structurally related compounds. In addition, it has been shown that liquid chromatography can be coupled to immunological assays. In this method, the sample is passed over an immunosorbent column that contains an antibody that is selective for the analytes of interest. After adsorption, the analytes are released from the immunosorbent column and subsequently separated on a second chromatographic In another method, fluorescently labeled antibody (Ab) is merged with antigens (Ag) after separation of the latter on a reverse phase microcolumn. The Ab@ complexes and free Ab are then passed through a column of immobilized antigen where only the flue rescent Ab:Ag complexes emerge and are detected.I5 While this system offers high sensitivity, it requires the use of a second column that needs to be regenerated frequently.

Because of the absence of a strong chromophore in the biotin moiety, and in order to improve the detection of biotin and its derivatives, precolumn derivatization techniques16-1s as well as postcolumn reaction detection systems based on the competitive binding p r i n ~ i p l e ~ ~ ~ ~ ~ have been developed. While these systems offer improvements in the detection limits over those of direct W absorbance detection, they are time consuming and/or do not have low enough detection limits for the direct determination of biotin in several samples including cell culture media. Recently, a fluorophore-linked protein binding assay using avidin labeled with fluorescein isothiocyanate (avidin-FITC) was coupled to HPLC to develop a postcolumn reaction detection system for biotin and biocytin.I4 This HPLC-BA system is based on an enhance- ment of the fluorescence intensity of avidin-FITC by the binding of biotin and biocytin. In a separate study, it was found that solutions of streptavidin-FITC gave greater fluorescence enhance- ment than avidin-FITC upon the binding of biotin and its

Przyjazny, A; Hentz, N. G.; Bachas, L. G. J. Chromatogr. 1993,654, 79- 86. Frutos, M.; Regnier, F. E. Anal. Chem. 1993, 65, 17A-25A. Desbene, P. L.; Coustal, L.; Frappier, F. Anal. Biochem. 1983, 128, 359- 362. Roder, E.; Englebert, U.; Traschutz, J. Freseniw' Z. Anal. Chem. 1984,

Stein, J.; Hahn, A; Lembcke, B.; Rehner, G. Anal. Biochem. 1992, 200,

Przyjamy, A; Kjellstrom, T. L.; Bachas, L. G. Anal. Chem. 1990,62,2536- 2540. Przyjazny, A; Bachas, L. G. Anal. Chim. Acta 1991, 246, 103-112.

0003-2700/95/0367-1014$9.00/0 0 1995 American Chemical Society

319, 426-427.

89-94.

derivatives.21 This allowed the development of batch-type assays for biotin using lower amounts of the labeled protein.

In this article, a homogeneous protein binding assay is used in a postcolumn reaction detection system for HPLC. The described HPLC-BA system combines the selectivity offered by HPLC with an additional level of selectivity and the enhanced sensitivity provided by the binding assay. Protein binding assays can discriminate among different families of compounds but sometimes cannot differentiate efficiently between structurally similar compounds. HPLC has the ability to separate structurally similar compounds but sometimes lacks the sensitivity necessary to detect analytes present in very low concentrations in complex matrices. However, when a homogeneous fluorophore-linked BA is used as a postcolumn reaction detection system for HPLC, an additional level of selectivity should evolve, where HPLC serves to separate the structurally related compounds and the BA selectively detects the analyte, thereby reducing or eliminating any signal due to interfering species. Thus, the coelution of sample constituents does not present a problem as long as any compounds that are structurally similar to the analyte(s) are separated by the HPLC; the separation need only be optimized for the structurally similar compounds and not for every compound in the sample. Indeed, if the analyte of interest coelutes with another structurally different compound, the latter will not be detected by the BA-based detection system. Finally, the described detection system was used to determine the biotin content of real samples, several of which contained biotin at levels below the detection limits of prior liquid chromatographic techniques, without the need for sample pretreatment or preconcentration.

EXPERIMENTAL SECTION Apparatus. All separations were carried out on a Rainin

Microsorb 5 p m CIS analytical column (250 mm x 4.6 mm). Immediately preceding the analytical column was a Rainin Mi- crosorb 5 p m CIS guard column (15 mm x 4.6 mm). The mobile phase was regulated by a Rainin Rabbit solvent delivery system (Rainin Instruments, Woburn, MA), and a Rheodyne Model 7125 injector with a 20-pL injection loop (Berkeley, CA) was used for sample introduction. For UV absorbance measurements, a Knauer Model 87 variable wavelength UV-vis absorbance detec- tor set at 220 nm was employed. The solvent delivery system and the UV-vis detector were interfaced to a Macintosh Plus computer (Apple Computer, Cupertino, CA).

Immediately following the absorbance detector, the effluent stream from the HPLC column was allowed to merge with the streptavidm-FITC reagent stream through a tee-connector (Figure 1). The postcolumn reagent stream was pumped by an Isco (Lincoln, NE) Model IC-2700 syringe pump. The two merging streams were allowed to mix in a 10.0-m knitted open-tubular reactor (prepared according to Selavka et aLZ2 ) made from FTFE tubing (0.5" i.d., 14" helix diameter).

Postcolumn reaction detection was carried out on a SPEX Fluorolog-2 spectrofluorometer (SPEX Industries, Edison, NJ) equipped with a 20-pL p-fluorescence flow cell (NSG Precision Cells, Farmingdale, NY). The excitation wavelength was set at 495 nm, while the emission was monitored at 518 nm. The emission and excitation slitwidths were each set at 2 mm. The

(21) Smith-Palmer, T.; Barbarakis, M. S.; Cynkowski, T.; Bachas, L. G. Anal.

(22) Selavka, C. M.; Jiao, IC-S.; Krull, I. S. Anal. Chem. 1987, 59, 2221-2224. Chim. Acta 1 9 9 3 , 2 7 9 , 287-292.

From Analytical Column

UV Detector at 220nm

H=~-N# Reagent Syringe Mixing Tee Reservoir

Reaction r-l Coil

Fluorescence I Detector

Back- Pressure Regulator

T 4

Waste

Figure 1. Schematic of the postcolumn reaction detection system.

SPEX spectrofluorometer was operated in the photon-counting mode. To prevent outgassing problems, a back-pressure regulator was placed after the fluorescence detector.

Reagents. Biotin, biocytin, monobasic sodium phosphate, and sodium bicarbonate (reagent grade) were obtained from Sigma (St. Louis, MO). Biotin ethylenediamine, Mbiotinoy1amino)- caproic acid, and Wbiotinoylamino) caproic acid hydrazide were purchased from Molecular Probes (Eugene, OR). HPLC-grade methanol was obtained from Baxter (Muskegon, MI), while acetonitrile was obtained from Aldrich (Milwaukee, WI). Strepta- vidin labeled with fluorescein isothiocyanate (FITC) (with 3.6 fluoresceins attached per streptavidin molecule) was obtained from Vector Laboratories (Burlingame, CA). Deionized (Milli-Q Water Purilication System; Millipore, Bedford, MA) distilled water was used to prepare all solutions. The mobile phase consisted of 80/ 20 (v/v) mixture of 0.100 M phosphate buffer (pH 7.O)/methanol that was pumped in an isocratic mode at a flow rate of 0.40 mL/ min.

M) were prepared by dissolving the compounds in a 0.100 M phosphate buffer solution (pH adjusted to 7.0). Working standards were made by further dilutions of the stock solution with the phosphate buffer. The streptavidin-FITC stock solution (purchased as 1 mg/mL solution) was diluted to a working 2 mg/L solution with 0.100 M phosphate buffer (pH 8.5). The streptavidin-FITC reagent solu- tions were prepared fresh daily unless otherwise specified.

Batch-Mode Optimization. To optimize the postcolumn reaction detection system, batch-mode experiments were con- ducted with disposable polystyrene cuvettes (Evergreen Scientific, Los Angeles, CA) using the SPEX spectrofluorometer (excitation at 495 nm and emission at 518 nm). Different concentrations of streptavidin-FITC in 0.100 M phosphate buffer (pH 8.5) were incubated with biotin standards (or buffer when measuring the background fluorescence intensity) for 5 min, after which the fluorescence intensity was measured. In some of the experiments, the effect of the presence of different concentrations of methanol and acetonitrile on fluorescence was also studied. Real Sample Analyses. To validate the postcolumn reaction

detection system, a number of real samples were analyzed for

Stock solutions of the analytes (4.0 x

Analytical Chemistry, Vol. 67, No. 5, March I, 1995 1015

their biotin content. Four different cell culture media, RPMI-1640 (RPMI), NCTC-135 medium (NCTC), Minimum Essential Me- dium Alpha (MEMA), and IPL41 insect cell medium (IPL) were purchased from Sigma. The NCTC, MEMA, and IPL media were prepared individually according to their accompanying instruc- tions. The RPMI medium was already in liquid form and therefore needed no sample preparation.

In addition, a sample of liquid infant formula (Isomil Ready- to-Feed Soy Protein Infant Formula, Ross Laboratories, Columbus, OH) was analyzed for its biotin content. The sample preparation was similar to that previously d e s ~ r i b e d . ~ ~ A 20-mL portion of the infant formula was denatured by addition of 150 pL of concentrated hydrochloric acid. The samples were then filtered through a Whatman No. 1 filter paper, and the denatured protein precipitate was rinsed with 1 mL of deionized distilled water. After the pH of the filtrate was adjusted to 7.0 with 6 M sodium hydroxide, the lipids and fats were extracted with four 8 m L portions of n-hexane. The aqueous portion was brought to a final volume of 25 mL with deionized distilled water.

RESULTS AND DISCUSSION In order to efficiently use binding assays as postcolumn

reaction detection systems, several general conditions have to be met. In particular, the interaction between the separated analytes and the binding protein should result in a change in an observable signal. The described HPLC-BA system Figure 1) uses an FlTC conjugate of streptavidin. The fluorescence emission intensity of the conjugate is enhanced when the binding sites of this labeled streptavidin are occupied by biotin or its derivatives. Batch-mode experiments were performed initially to determine the optimum conditions that yield maximum enhancement of fluorescence intensity. This optimization study included the streptavidin-FITC concentration and the mobile phase organic modifier composition. Streptavidin-FITC concentrations were varied between 1.0 and 3.0 mg/L. Concentrations above 2.0 mg/L streptavidin-FITC gave fluorescence intensity enhancements -10% higher than that produced by 2.0 mg/L. Therefore, in an effort to keep the cost of analysis at a minimum, the streptavidin-FITC concentration of 2.0 mg/L was chosen for subsequent studies.

Using a streptavidin-FITC concentration of 2.0 mg/L, the effects of methanol and acetonitrile on fluorescence enhancement were evaluated. Methanol and acetonitrile were both examined because they had been previously used as mobile phase organic modifiers in HPLC systems for biotin and bi~cytin.'~J* Methanol produced fluorescence enhancements equal to or greater than those produced by acetonitrile for each concentration of organic modifier studied. Thus, the use of acetonitrile as a possible organic modifier was not pursued further because of its slightly greater quenching effects, its cost, and its potential toxicity.

Methanol was further examined to determine whether the previously established separation conditions for biotin and its derivatives (Le., 80% aqueous buffer/20% methanol (v/v)14) would alter the fluorescence intensity. Taking into account the dilution of the mobile phase after it merges with the streptavidin-FITC reagent stream, streptavidin-FITC actually interacts with an approximately 7% (v/v) methanol solution. Batch-mode experi- ments were performed in which the biotin-induced fluorescence enhancement of streptavidin-FITC was examined in the presence

(23) Nicolas, E. C.; Pfender, K A. J Assoc. Ofi Anal. Chem. 1990, 73, 792- 798.

Biotin Concentration (M)

Figure 2. Batch-mode experiment showing the effect of methanol on fluorescence enhancement. The concentration of streptavidin-FITC remained constant at 2.0 mg/L. In each cuvette, 0.20 mL of biotin standard was added to 2.80 mL of the streptavidin-FITC/methanoC containing buffer (pH 7.0). Methanol contents of 1% (+), 3% (A), 6% (0), 10% (x) , 20% (m), and 50% (a) were examined.

of different amounts of methanol. As shown in Figure 2, solutions containing 1%- 10% methanol gave similar fluorescence enhance- ment. However, significant differences in fluorescence enhance- ment were observed when the methanol content increased to more than 10%. These results suggested that the existing conditions for the separation of biotin and its derivatives would not affect the overall fluorescence intensity enhancement produced by the binding of biotin to streptavidin-FITC,

Once the optimum streptavidin-FITC concentration and amount of organic modifier were determined by batch-mode experiments, the effect of reagent flow rate in the postcolumn reaction detection system of Figure 1 was examined. The optimum flow rate of the streptavidin-FITC reagent was determined by varying the flow rate from 0.05 to 2.5 mL/min using a syringe pump and 4 x M biotin and biocytin standards. It was determined that a flow rate of 0.10 mWmin gave the greatest peak area while maintaining baseline resolution between biotin and biocytin. According to Selavka et a1.,22 the knitted open-tubular reactor coil has advan- tages (e.g., less band broadening) over a linear open-tubular reactor only at higher flow velocities. While this was found to be true, there was also an increase in background fluorescence at higher flow rates. Therefore, a reagent flow rate of 0.10 mL/min was chosen over the higher flow rates because of the higher signal-tmoise ratio, even though there was greater band broaden- ing. At flow rates lower than 0.10 mL/min, baseline resolution was sacrificed for a greater signal-to-noise ratio.

The stability of the streptavidin-FITC reagent was also deter- mined. It was found that under our experimental conditions, the streptavidin-FITC reagent was stable for at least 96 h. The stability was examined by letting the same streptavidin-FITC reagent remain in the syringe pump while periodically running chromato- grams and subsequently measuring the chromatographic peak area that corresponded to the same 4 x M biotin standard. There was no observable decrease in peak area over this 9&h time period. The previously reported avidin-FITC postcolumn reaction detection system showed reagent stability of only 8 h.I4 This is not surprising given the well-known higher nonspecific adsorption demonstrated by avidin in several application^.^^ This nonspecific adsorption has been ascribed to avidin being a glycoprotein. Streptavidin is not a glycoprotein and therefore is adsorbed to a lesser extent on the walls of the syringe pump.

1016 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995

Table 1. Comparison between the Biotin Contents of Four Cell Culture Media Determined by the Proposed System and Those Claimed by the Manufacturer

biotin" biotin foundb sample Cug/mL) Cug/mL) RSDC

RPMI 0.2 0.19 f 0.01 5.2 NCTC 0.025 0.027 f 0.001 3.7 MEMA 0.1 0.100 f 0.001 1.0 IPL 0.16 0.166 f 0.001 0.6

a Biotin content as claimed by the manufacturer. Biotin content found by the described postcolumn reaction detection system, along with the standard deviation (n = 3). Relative standard deviation (%).

0 $ 130000- c

0.0 5,; 10.0 15.0 19,o Time (min)

u 2200004 8

" - c = 200000 .- E le0000 - 8 160000 8 n 140000

Biotin

w f G 120000

0.0 5.0 10.0 15.0 19.0 Time (min)

Figure 3. (A) Chromatogram of IPL-41 insect cell culture medium using UV absorbance detection at 220 nm. The y-axis signal is measured in millivolts and corresponds to absorbance. (B) Chro- matogram of the same IPL-41 insect cell culture medium using fluorescence detection. Mobile phase, 80/20 (v/v) mixture of 0.100 M phosphate buffer (pH 7.0)lmethanol; mobile phase flow rate, 0.40 mumin; postcolumn reagent, 2.0 mg/L streptavidin-FITC in 0.100 M phosphate buffer (pH 8.5); postcolumn reagent flow rate, 0.10 m U min. The postcolumn reaction was allowed to take place in a 10.0-m knitted open-tubular reactor.

Calibration plots were constructed for both biotin and biocytin by plotting peak area versus the biotin (or biocytin) concentration. The linear range of the calibration plot extended from 8 x to 1 x M for both biotin and biocytin. The minimum detectable peak, with a signal-to-noise ratio of 3, for biotin and biocytin was found to correspond to a concentration of 2 x M. This is equivalent to 97 and 149 pg of biotin and biocytin, respectively. It was also determined that there was some additional peak broadening as a result of the postcolumn knitted open-tubular reactor with respect to the W absorbance chro- matogram. However, baseline resolution between biotin and biocytin was still possible under the indicated chromatographic conditions.

To examine the analytical utility of the described system, a variety of samples were analyzed for their biotin content. Figure 3 represents a typical chromatogram of the IPL41 insect cell culture medium. The difference in retention times between parts A and B is due to the introduction of a 10.@m knitted open-tubular reactor, which was necessary to facilitate the association reaction between biotin and streptavidin-FITC. It should also be noted that the corresponding chromatograms in parts A and B are of the same sample injection using W absorbance (part A) and fluorescence detection (part B) in series. The biotin content determined in the four cell culture media agreed very well with the values claimed by the manufacturer (Table 1). It should be noted that these cell culture media are complex and contain a variety of salts, amino acids, and vitamins. For example, the IPL 41 insect cell culture medium contains 21 amino acids, 11 vitamins,

0 5 t'0 i5 20 Time (min)

t 5 140000{

Biotin n

sugars, and several inorganic salts. No sample pretreatment other than filtration was necessary.

A fifth sample (Isomil infant formula) was also analyzed for biotin using the same postcolumn reaction detection system. After the biotin was extracted from the infant formula, the prefiltered sample was injected straight with no preconcentration or dilution step. The amount of biotin in the infant formula was determined to be 0.028 f 0.001 ,ug/mL (n = 3), in good agreement with the manufacturer's claim of 0.030 yg/mL. The relative standard deviation was 3.5%. Atypical chromatogram for the infant formula extract is shown in Figure 4, where Figure 4A represents the W absorbance detection and Figure 4B represents the fluorescence postcolumn reaction detection.

Besides the sensitivity and accuracy of this homogeneous fluorophore-linked postcolumn reaction detection system, the system also has excellent selectivity. The selectivity offered by the homogeneous fluorophore-linked protein binding assay can be illustrated by the typical chromatograms in part B of Figures 3 and 4. Because of the W-absorbing species present in the various samples, the W absorbance chromatograms (part A in

Analytical Chemistry, Vol. 67, No. 5, March 1, 1995 1017

‘ 1 i

0 10 20 30 Time (min)

Figure 5. Chromatogram of a 20-pL injection of a standard containing 8 x lo-’ M biotin ethylenediamine ( l ) , biotin (2), biocytin (3), 6-(biotinoylamino)caproic acid (4), and 6-(biotinoylamino)caproic acid hydrazide (5). The postcolumn reagent flow rate was 0.50 mU min, while all of the other chromatographic conditions remained the same as previously described.

Figures 3 and 4) show several peaks. The peak at approximately 14 min in Figure 3 (part A) represents the vitamin niacinamide, which coelutes with biotin under these chromatographic condi- tions. It should also be noted that while niacinamide and biotin coeluted, the biotin in the sample is present at concentrations at or below the limit of detection using direct W absorbance detection at 220 nm. Thus, quantification of biotin by direct W absorbance at these concentrations would be highly unreliable even when the biotin peaks could be measured above background. However, niacinamide neither induces a fluorescence enhance- ment in streptavidin-FITC nor fluoresces under the conditions utilized in the postcolumn reaction detection system. Using streptavidin-FITC, the chromatograms shown in part B (Figures 3 and 4) were obtained, where the peaks due to biotin are easily detected. No interference by niacinamide was observed when the postcolumn reaction detection system was used. Further, because of the selectivity of the homogeneous BA, the interfering signal from other vitamins and sample components is virtually eliminated.

Figure 5 demonstrates that the described HPLC-BA system responds to the class of compounds that contain the biotin moiety. Biotin, biocytin, biotin ethylenediamine, &(biotinoyIamino) caproic acid, and &(biotinoylamino)caproic acid hydrazide induce an enhancement in the fluorescence intensity of streptavidin-FITC.

(25) Mock, D. M.; Lankford, G. L.; Cazin, J. J. Nutr. 1993, 123, 1844-1851. (26) Haugland, R. P. In Molecular Probes: Handbook of Fluorescent Probes and

Reseerch Chemicals; Larison, K. D., Ed.; Molecular Probes: Eugene, OR, 1992; pp 62-65.

Because all of the above mentioned compounds would increase the fluorescence intensity of streptavidm-FITC, direct quantifica- tion of one of the components by a binding assay is not feasible. However, the chromatographic peaks are resolved (Figure 5) under the chromatographic conditions described in the figure legend. Note that biotin eluted in less time than in Figures 3 and 4 since the reagent flow rate was higher. This higher reagent flow rate also accounts for an increase in the fluorescence background as well as the sharper peaks. It should also be noted that while biotin and biocytin are the major biotin derivatives found in natural biological systems,2j the synthetic derivatives biotin ethylenediamine, &(biotinoylamino)caproic acid, and &(biotinoy- 1amino)caproic acid hydrazide are commonly used as protein modification reagents.26 Therefore, the developed liquid chro- matographic system could be employed to detect biotin derivatives other than the naturally occurring biotin and biocytin.

The streptavidin-FITC postcolumn reaction detection system ($0.30 per analysis) has a cost advantage over the previously reported avidin-FITC postcolumn reaction detection system ($0.50 per analysis) while maintaining similar detection limits.14 The cost of analysis was based on the postcolumn reagent used (Le., streptavidin-FITC or avidin-FITC) .

A number of advantages resulted from using this postcolumn reaction detection system. First, little or no pretreatment of the real samples was necessary. Second, a preconcentration step that would have been required for direct UV absorbance detection of biotin was not necessary since fluorescence detection offers lower detection limits. Finally, the described postcolumn reaction detection system required no optimization with respect to resolu- tion between biotin and other coeluting interferents (e.g., amino acids, vitamins, carbohydrates, etc.). Only the resolution among biotin and its derivatives needed to be optimized. Thus, HPLC- binding assay systems offer several advantages over either HPLC or binding assays alone.

ACKNOWLEDGMENT This research was supported by grants from the National

Science Foundation (Grant CTS-9307518) and American Cyana- mid.

Received for review September 19, 1994. Accepted November 29, 1994.@

AC940937G

@ Abstract published in Advance ACS Abstracts, January 1, 1995.

1018 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995