CHROMATOGRAPHIC SEPARATION AND i.:‘ FLUORDMETRIC DETERMINATION OF _ :PYRIDOXAL, PYREDOXAMINEAND _ . ‘ y _ ~ PYREDEJXIN'EEN F909? SYSTEM . . ,E w .1 f : *' i ; Ehesis fay Ehe Degree of M S. _ . MECHEW SEATE UNWERSEEY EYEEE PENGCHENU ' gigifi ;. ..... ABSTRACT PROCEDURE FOR CHROMATOGRAPHIC SEPARATION AND FLUOROMETRIC DETERMINATION OF PYRIDOXAL, PYRIDOXAMINE AND PYRIDOXINE IN FOOD SYSTEMS By YEN-PING CHIN Various procedures have been developed for the separation and determination of vitamin 36' Although no one assay procedure has been satisfactory for the determination of all three forms of vitamin B6’ the microbiological assay using Saccharomyces carlsbergensis has been widely used for measuring the vitamin B6 content in foods. The fluorometric procedure reported here is a com- bination and modification of numerous chemical methods for the determination of vitamin B6. Separation of pyridoxal, pyridoxamine and pyridoxine was carried out by Dowex AG 50 ion exchange chromatography. Quantitation of these purified fractions was accomplished by fluorometry following their conversion to 4-pyridoxic acid lactone. Pyridoxal was con- verted to 4-pyridoxic acid lactone using potassium.cyanide. Pyridoxine and pyridoxamine were first converted to pyridoxal by manganese dioxide and sodium glyoxalate, respectively, and then oxidized to 4-pyridoxic acid lactone. Fluorescence Yen-Ping Chin 'was measured at 355 nm (excitation) and 436 nm (emission) at pH 9610. Selected food samples were chosen to evaluate the applicability of the fluorometric method for the determina- tion of vitamin B6 in foods. The microbiological method of Toepfer et a1. (A.0.A.C., 1961) was used as a basis of comparison for the proposed fluorometric method. Recovery values for the three vitamin B6 fractions varied for each food sample assayed. The percent recovery of pyridoxal and pyridoxamine for the selected foods using the fluorometric assay varied between 83 to 110 with the standard deviation ranging from 2 to 13. The percent recovery for the same food products analyzed by the microbiological method exhibited a range of 50 to 110 with the standard deviation ranging from 7 to 23. Using the fluorometric assay the recovery values for pyridoxine from the selected foods varied from 55 to 74 with standard deviation ranging from 3 to 7, while the recovery values for the microbiological method varied from 99 toJ32 with standard deviation ranging from 8 to 31. Comparisons of total vitamin B6 values for various food products indicated that values obtained by the fluoro- metric method exhibited almost twice as much total vitamin B6 as those obtained by the microbiological method. CHROMATOGRAPHIC SEPARATION AND FLUOROMETRIC DETERMINATION OF PYRIDOXAL, PYRIDOXAMINE AND PYRIDOXINE IN FOOD SYSTEM BY YEN— P ING CHIN A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Dept. of Food Science & Human Nutrition 1975 ACKNOWLEDGEMENT The author expresses her sincere gratitude to her adviser, Dr. James Kirk, for his patience and counsel during this study and for his constructive criticism of the manu- script. Special appreciation is also extended to Dr. Kenneth Stevenson for his guidance and assistance in the micro- biological assays. The author also wishes to express her thanks to her committee members, Dr. Kenneth Stevenson and Dr. Richard Luecke for reviewing the manuscript. Grateful acknowledgement is due to the Department of Food Science, Michigan State University, for the facilities and funds which make this research possible. Grateful acknowledgement is also due to Mrs. Margeurite Dynnik for her technical assistance in the micro- biological analyses. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS . LIST OF TABLES . LIST OF FIGURES INTRODUCTION . LITERATURE REVIEW Vitamin B : History . Vitamin.Bg: Chemical and. PhysicaI Properties. Vitamin BG: Methods of Determination Microbiological Methods . Extraction of Vitamin B6 Acid Hydrolysis . . Enzymatic Hydrolysis Column Chromatography . Chemical Methods Colorimetric . Spectrophotometric. Fluorometric . Lactone Method . . . Cyanohydrin Method Biological Assays . METHOD . . . . . . . . Reagents for Chemical and Micrdbiological Determination . . Apparatus for Fluorometric Determination Samples used for A.na1ysis . Sample Preparation for Fluorometric Deter-o mination of Vitamin B6 Pyridoxal . . Pyridoxamine Pyridoxine iii Page ii vii viii \OCh Uh>w w |'--| . 13 . 16 . 16 . 17 18 18 '. 20 .24 .27 .27 .28 .28 .29 .30 :30 TABLE OF CONTENTS (Continued . . .) Page Standards . . . . . . . . . . . . . . . . . 31 Recovery Standards . . . . . . . . . . . . 32 Continuous Flow Analysis . . . . . . . . . . . 32 Calculations . . . . . . . . 33 Microbiological Assay for Vitamin B6 . . . . . . . 33 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 35 Extraction . . . . . . . . 35 Separation of Pyridoxal, Pyridoxamine, and Pyridoxine by Column Chromatography . . . . . . . 35 Fluorometric Determination of Pyridoxal, Pyridoxamine and Pyridoxine Standards . . . . . . 36 Microbiological Determination of Pyridoxal, Pyridoxamine and Pyridoxine Standards . . . . . . 36 Vitamin B6 Contents in Selected Food Products . . . 37 Pyridoxal . . . . . . . . . . . . . . . . . . . 37 Pyridoxamine . . . . . . . . . . . . . . . . . 37 Pyridoxine . . . . . . . . . . . . . . . . . . 38 Comparisons of Fluorometric and Microbiological Total Vitamin B6 Values . . . . . . . . . . . . . 38 Sample Blanks . . 39 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 53 Extraction . . . . . . . . . . . . . . . . . . . . 53 Enzymatic Extraction . . . . . . . . . . . . . 53 Acid Extraction . . . . . . . . . . . 54 Limitation of Acid Hydrolysis . . . . . . . . . 55 Colum Chromato otgraphy . . . - - . 57 Function 0 the Iondexchange Column . . . . . . 57 Criticism.of the Ion-Exchange Column . . . . . 58 Fluorometric Determination of Pyridoxal, Pyridoxamine, and Pyridoxine Standards . . . . . . 59 Mechanism of Reactions . . . . . . 60 Analysis of the Blanks for the Vitamin B6 Standards . . Microbiological Determination of Pyridoxal, Pyridoxamine and Pyridoxine Standards . . . . . . 61 Microbiological Determination of Vitamin B6 in . 61 Selected Food Products . . . . 62 Fluorometric Determination of Vitamin B6 in Selected Food Products . . . . . 63 iv TABLE OF CONTENTS (Continued . .4.) Page Ham . . . . . . . . . . . . . . . . . . . . . . 63 Infant Formula . . . . . . . . . . . . . . . . 64 Cereal . . . . . . . . . . . . . . . . . . . . 64 Lima Beans . . . . . . . . . . . . . . . . . . 65 Analysis of Sample Blank . . . . . . . . . 66 Interferring Substances in the Fluormmetric Determination of Vitamin B6 . . . . . . . . . . . . 67 Nicotinamddeadeninedinucleotide . . . . . . . . 67 Metal Ions . . . . . . . . . . . . . . . . . . 68 Manganese Dioxide . . . . . . . . . . . . . . . 68 Thiamin . . . . . . . . . . . . . . . . . . . . 69 Comparison of Vitamin B6 Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods . . . . . . . . . . . . . . 69 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . 72 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 74 Table 11a. 11b. IIIa. IIIb. IVa. IVb. VI. LIST OF TABLES A Comparison of Enzymatic and Acid Hydrolysis for the Determination of Vitamin B6 in Infant Formula . Pyridoxal Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods . Pyridoxal Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods . . Pyridoxamine Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods . . Pyridoxamine Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods . . . . Pyridoxine Values in Selected Food Samples Determined by the Fluorometric and Microbiological Methods Pyridoxine Values in Selected Food Samples Determined by the Fluorometric and Microbilogical Methods . . Comparison of Total Vitamin B Values for Selected Food Products Using the Microbiological and Fluorometric Methods . Study of Sample Blanks Using the Fluorometric Method . . . . vi Page . 41 . 42 . 43 . 44 . 45 . 46 . 47 . 48 . 49 Figure 1. LIST OF FIGURES Standard Curves for Pyridoxal, Pyridox- amine, and Pyridoxine Using Fluorometric Method . . . . . . . . . . . . . . . . . Standard Curves of Pyridoxal, Pyridoxamine and Pyridoxine Using Micrdbiological Method . . . . . . . Comparison of Fluorescence Response for Sample and Sample Blank During Pyridoxal Assay of Selected Food Samples . . Continuous Flow Automated Analysis System for Determining Vitamin B6 in Foods vii Page . 50 . 51 . 52a . 52b INTRODUCTION Pyridoxal, pyridoxamine and pyridoxine, the three biologically active forms of vitamin 36’ have been shown to occur in the free form or combined with phosphate groups or with proteins. Although numerous procedures have been developed for the separation and determination of the various forms of vitamin B6, no single assay procedure has been found satisfactory for the quantitative determination of vitamin B6 in foods. The Saccharomyces carlsbergensis microbiological assay developed by Atkin et a1. (1943), is generally the recognized method for the determination of total vitamin B6 in foods and biological materials, even though it does not respond equally to the three biologically active forms of vitamin B6. Chemical methods utilizing colorimetric, spectro- photometric and fluorometric techniques have been found satisfactory with relatively pure and concentrated solutions of vitamin B6' Attempts to adapt these chemical methods to the measurement of vitamin B6 in foods have not been successful, because of interferring substances within the food products. The purpose of this study was to develop a chemical method for the quantitative determination of vitamin B6 in 1 2 food products and to reduce the time required for analysis. The S. carlsbergensis microbiological method was used as a basis of comparison for the proposed chemical procedure. LITERATURE REVIEW Vitamin B6: History The existence of vitamin B6 as a dietary essential was first recognized by Gyorgy in 1936. The isolation and identification of pyridoxine from several natural sources facilitated the elucidation of its structure and chemistry, which led directly to its synthesis (Lepkovsky, 1938; Keresztesy and Stevens, 1938; Gyorgy, 1938; Kuhn and Wendt, 1938; Ichiba and Michi, 1938; Harris and Folkers, 1939). The terms "vitamin B6 and "pyridoxine" were synonymous until Snell et a1. (1942) found two related sub- stances in food materials that surpassed pyridoxine in growth-promoting activity for certain species of lactic acid bacteria. Further work by Snell (l944a,b) and Harris et al. (1944a,b) demonstrated that the related substances were pyridoxamine and pyridoxal. Concurrent with these studies, research by other workers indicated that vitamin B6 functioned as a coenzyme in the amino acid metabolism of lactic acid bacteria (Gale, 1944; Bellamy and Gunsalus, 1944). This led to the dis- covery of the metabolically active phosphorylated forms of the vitamin; pyridoxal phosphate and pyridoxamine phosphate (Gunsalus and Bellamy, 1944; Heyl et al., 1951; Baddiley and 3 4 Mathias, 1952; Rabinowitz and Snell, 1947). Vitamin B6: Chemical and Physical Properties The various forms of vitamin B6 are widely distri- buted in a large number of foods of animal and plant origin. The major portion, existing in the free form, is associated primarily with protein and starch. The naturally occurring forms of vitamin B6 differ only by the substitute groups at the C-4- position. CHZOH /' HCLCNO: N H3C Pyridoxin Pyridoxal Pyridoxamine Pyridoxine (PIN), pyridoxal (PAL), and pyridoxamine (PAM), which are colorless and odorless crystals, exist as free bases or as the commonly available hydrochlorides. PAL, PAM, PIN are resistant to heat and acid but are decomposed by alkali, ultraviolet light, and oxidizing agents, such as nitric acid, potassium permanganate, and hydrogen peroxide (Cunningham.and Snell, 1945). The three biologically active forms of vitamin B6 exhibit different light absorption maxima, as well as, different fluorescence characteristics. These latter two properties were the basis for several chemical methods of 5 analysis, which will be discussed later in detail. Vitamin B6: Methods of Determination Pyridoxal, pyridoxamine and pyridoxine, which ex- hibit equal biological availability for man and higher animals, have been shown to occur in the free form or com- bined with phosphate groups and/or proteins. Thus, the estimation of total vitamin B6 in foods requires the quanti- tative determination of these three components. No single assay procedure has been reported satis- factory for the determination of all three forms. Thus, procedures for the separation and determination of the active forms of vitamin B6 were essential to the development of a quantitative vitamin B6 assay. Methods available for the determination of vitamin B6 were divided into four categories; microbiological, physical, chemical and animal.. Microbiological procedures were the preferred method for determining the vitamin B6 content of foods and biological materials. However, each species of microorganism responded differently to the various forms of vitamin B6. This made it difficult to evaluate and compare assay results using different micro- organisms. Physical and chemical methods were satisfactory 'with only relatively pure and concentrated vitamin B6 preparations. The rat growth assay, on the other hand, gave erroneously high values if the samples analyzed contained growth promoting factors in addition to vitamin B6. Microbiological Methods Microbiological methods have been used more fre- quently than any other method in assaying foods and bio- logical materials for vitamin B6. These methods employed microorganisms such as lactic acid bacteria (Rabinowitz and Snell, 1947), yeast (Atkin et al., 1943) and a mold mutant (Stokes, 1943). Since the three forms of vitamin B6 could elicit different activities for different microorganisms, the following three factors should be evaluated before choosing a method of assay: the form of the vitamin to be determined, the microorganism.which would give the maximum response, and the material being assayed. To date, no unqualified recommendations have been ‘made for any one microbiological assay method for vitamin B6 in complex biological substances. This is primarily due to the inherent nature of the assay organismsy and the in- adequacies of extraction procedures. The Saccharomyces carlsbergensis assay, developed by Atkin et a1. (1943) and modified by WOodring and Storvick (1960), had been generally accepted for the estimation of total vitamin B6. The assay, based on the growth of the microorganism, was measured turbidimetrically. It was chosen for its simplicity, convenience, speed, and the ability of S. carlsbergensis to respond similarly to all three forms of vitamin B6. However, Parrish et a1. (1955) 7 demonstrated that on occasion certain strains of the organism gave lower response to pyridoxamine than to pyridoxine or pyridoxal. Rabinowitz and Snell (1948) developed a differential technique for assaying the three components of vitamin B64 as well asg’total vitamin 36' Their procedure was based on the I observation that Streptococcus faecalis R. responded to pyridoxal and pyridoxamine; Lactobacillus gaggi responded only to pyridoxal; and S. carlsbergensis responded to all three forms of the vitamin. Individual forms of vitamin B6 were determined by subtraction. However, the pyridoxine value obtained from this differential method was probably higher than its actual content in the product, due to the inability of any organism.to measure pyridoxine and the lack of sensitivity of S. carlsbergensis toward pyridoxamine. Gregory (1959) reported that the differential assay technique of Rabinowitz and Snell (1948) was improved by the use of S. faecium 9 51. This organism did not respond to DL-alanine or D-alanine, which was shown to replace pyridoxal or pyridoxamine as a growth factor for S. faecalis R. The primary function of vitamin B6 in _S_. faecalis R. appeared to be that of a coracemase in the conversion of L- to D- alanine. Therefore, if D-alanine was present in the medium, time requirement for the vitamin was eliminated. DL-alanine, hcnwever, was not shown to replace pyridoxal or pyridoxamine as a growth factor for S. faecium.¢ 51. 8 Diding (1955) proposed a method utilizing a bio- chemical mutant of Escherichia coli, strain 154-59 L, for the determination of total vitamin B6. This procedure offered the advantage of simplicity with regard to medium preparation) but was limited to assaying pure compounds, such as; multivitamin preparations. Baker et a1. (1962, 1963) proposed a method using Tetrachymena pyriformis, a protozoan, for the determination of vitamin B6 in tissue, urine, blood, serum, and cere- brospinal fluid. This procedure was slower to perform than the §_. carlsbergensis method, requiring an incubation period of 5 days. The T. pyriformis method was also limited by the fact thatglpxpyridoxine appeared to have considerably less activity than pyridoxamine on the assay organism. Thus, significant errors would have been encountered in using this organism for the assay of vitamin B6 content of plant products or other materials that contained an appreciable amount of pyridoxine. A plate method using the mold mutant, Neurospora gitophila, was described by Haenel and Muller-Beuthow (1956) . Although not sensitive, this method had the advantage of requiring only a six hour incubation period. An additional method utilizing the mold mutant, H- sitophila, received limited attention for the determina- tion of total vitamin B6 in biological materials (Stokes et al. , 19 43). Although the organism appeared to respond equally 9 well to all forms of the vitamin, the assay was complicated, time consuming, and required an incubation period of 5 days. Extraction of Vitamin B6 An adequate extraction method for the liberation of vitamin B6 from its multiple bound forms has not been reported. To some degree this problem has been shown to account for many of the discrepancies in vitamin B6 values reported in foods and biological materials, especially blood and tissues. Birch and Gyorgy (1936) first noted the bound state of vitamin B6' Siegel et a1. (1943), using acid hydrolysis, demonstrated the necessity for the liberation of these bound forms for accurate chemical or microbiological assays. Since 1943 many diverse types of hydrolysis procedures were developed. Generally three types of hydrolysis; acid hydrolysis, enzymatic hydrolysis, and the combination of acid and enzymatic hydrolysis, have been employed to release bound vitamin 36' Acid Hydrolysis Atkin et a1. (1943) found that efficient acid ex- traction depended not only upon the strength of the acidx but also upon the volume of acid used. They recommended 'using a sample which contained the equivalent of 2-4,ug of inttamin B6 and hydrolyzing in 180 ml of 0.055 N H2804 for .1 liour at 20 psi steam. Later, many investigators modified Atkin's method by changing both the concentration of acid and 10 the length of hydrolysis. Rabinowitz and Snell (1948) improved the extraction method of Atkin et a1. (1943) by in- creasing the hydrolysis time from 1 hour to 5 hours, the pressure from.15 psi to 20 psi and substituting H2804 for HCl. Fujita et a1. (l955a,b) reported that pyridoxine and pyridoxamine were best extracted by preheating the sample homogenate, pH 4.5, at 80°C for 15 minutes, adjusting the supernatant to 0.6 N H2804 with 3 N H2804 and autoclaving at 1300 C for 1 hour. However, better results were obtained for pyridoxal and 4-pyridoxic acid when the homogenate was heated directly in 0.1 N H2804 at 1300 for 1 hour. In 1959, Gregory demonstrated that samples heated in 0.55 N HCl under steam for 30 minutes gave results which were similar to those obtained by autoclaving and adopted this process for extraction. Benson et a1. (1964) studied the effect of hydroly- sis time on vitamin B6 recovery using 0.055 N HCl and 15 psi. Hydrolysis time was varied from 30 minutes to 72 hours. The results showed that both L, ggggi and S. faecium.increased in growth rate after 30 minutes of hydrolysis and reached a peak between 10 and 24 hours. After 24 hours, growth decreased until leveling off at 72 hours at approximately the same value obtained between 2 and 5 hours. Tests with {§. carlsbergensis showed significantly different results. The highest value was obtained after 2 hours of hydrolysis. A slight decrease was observed from 2 to 5 hours. Followed 11 by a sharp decrease to an almost unreadable minimum after 10 hours. Another vitamin B6 extraction method required the removal of protein from the sample with trichloroacetic acid or perchloric acids, however, this resulted in the removal of the phosphorylated and protein bound vitamin B6 complex from the sample extract. Storvick and Peters (1964) studied this phenomenon and reported that blood subjected to acid hydrolysis and assayed with S, carlsbergensis, gave vitamin B6 values 1 to 1% times higher than the protein free filtrate. Enzyme Hydrolysis Most of the vitamin B6 in food and biological materials has been found to be tightly bound to protein (Birch and Gyorgy, 1936). Therefore, it was advisable to hydrolyze protein rich materials)such as, meat and blood,be- fore acid extraction. This prevented occlusion of the vitamin in the protein precipitate resulting from.heat de- naturation. Preliminary protein breakdown was usually accomplished with papain, pepsin, Taka-diastase, and alkaline or acid phosphatase. The utilization of enzymes for the extraction of vitamin B6 was for the most part unsatisfactory, since many of the enzyme preparations contained a considerable amount of vitamin B6 (Rabinowitz and Snell, 1947; Hopkins and Pennington, 1947). 12 Williams et a1. (1942) reported low vitamin B6 values for milk using enzymatic hydrolysis with microbiological assay methods. Siegel et a1. (1943) postulated that the value obtained by Williams et al. (1942) represented only free vitamin B6 and not bound vitamin B6' Swaminathan (1940) reported higher vitamin B6 values for milk using a combina- tion of pepsin digestion and acid hydrolysis before chemi- cal determination. Gregory and Mabbitt (1961) reported pyridoxamine phosphate was more resistant to hydrolysis than pyridoxal phosphate. In both milk samples and standard solution alkaline phosphatase was used after acid hydrolysis to achieve complete release of pyridoxamine from pyridoxamine phosphate. Storvick and Peters (1964) reported that samples subjected to prolonged heat treatment exhibited a decrease in microbial growth. It was theorized that this decrease was a result of a rebinding or condensation of free vitamin B6 to protein or phosphate groups, which rendered the vitamin biologically unavailable. This theory was supported by the results of Storvick and Peters (1964) who reported no destruction of standard solutions of pyridoxal, pyridox- amine and pyridoxine after 72 hours of acid hydrolysis. However, when milk was subjected to the same hydrolytic con- ditions, a decrease in the vitamin B6 content of the milk resulted after 5 hours of autoclaving. In this same report, 13 a recovery study was performed in which standard solutions of vitamin B6 were added to both.mdlk and blood prior to hydrolysis. They found that in the presence of protein, prolonged heat treatment resulted in losses of vitamin B6. Results from.this study supported the findings of Fujita et a1. (l955a,b), who reported that maximum.liberation of vitamin B6 depended on acid concentration, pressure, and the length of hydrolysis. Fujita et a1. (l955a,b) further suggested that strong acid, high pressure and short extraction time ‘would provide the best conditions for the liberation of vitamin B6 and minmmized rebinding. To date, no method is available for determining if vitamin 36 is totally liberated following acid hydrolysis. Therefore, the hydrolysis procedure which would result in the highest vitamin B6 recoveries was used as a guideline for determining the completeness of hydrolysis. Storvick et a1. (1964) further suggested that acid hydrolyzates should be subjected to treatment with acid or alkaline phosphatase to determine if vitamin B6 was hydrolyzed from its phosphorylated forms. Column Chromatography Storvick and Peters (1964) reported that biological materials could contain substances which were inhibitory to vitamin B6 assay organisms. They found that a 20-hour hydrolyzate of blood, which had no vitamin B6 activity for g. carlsbergensis, gave a value of approximately 25 ng per l4 ‘milliliter of blood after the hydrolyzate had been passed over Dowex 50 (Kf) column. This substantial increase indi- cated that a substance inhibitory to the yeast had been removed from the hydrolyzate by ion-exchange chromatography. Peterson and Sober (1954) reported the first com- plete column chromatographic separation of pyridoxal, pyridoxamine, pyridoxine, the phosphate forms, and 4- pyridoxic acid. A weak cation exchange resin, Amberlite XE-64 (H+), 'with water as the eluant was used for the separation of pyridoxal-5'-phosphate, pyridoxic acid and pyridoxamine-5'-phosphate. .Acetic acid (5%) was used to elute pyridoxal, pyridoxine, and pyridoxamine. Fractions were collected and examined by spectrophotometry and paper chromatography. This procedure required high concentrations of pure vitamin B6 and did not prove satisfactory for separat- ing small amounts of the vitamin B6 fractions from biologi- cal materials containing significant levels of interfering substances. Fujita et al. (1955a,b) proposed a procedure for the chromatographic separation of the various components of vitamin B6 present in biological materials. The various forms of B6 were recovered as follows: 4-pyridoxic acid, Amberlite IRA-410 (Ac‘); pyridoxine, Permutit; pyridoxal, IRC-50 (H+) or IR-112 (H+). Pyridoxamine was deaminated to pyridoxine, which was then adsorbed on a Permutit column. Although their method was complicated, it represented the first successful attempt to isolate pure vitamin B6 fractions 15 from biological materials and was fundamental to the development of separation schemes in recent years. A less complicated method using Dowex AG 50W2X8 and differential elution was proposed by Toepfer and Lehman (1961) for the separation of the three forms of vitamin B6. Pyridoxal, pyridoxine, and pyridoxamine were eluted with 100 m1 aliquots of 0.04 potassium acetate, pH 6.0; 0.1 M potassium acetate, pH 7.0; and 1.0 M KCl-0.l MIKZHPOA, pH 8.0, respectively. Eluates were assayed microbiologically using §. carlsbergensis, however, it was not possible to determine whether complete separation had been attained, because S. carlsbergensis could not differentiate the three forms of the vitamin. Later, studies by Storvick et a1. (1964) indicated that distinct separation of the components of vitamin B6 was not achieved with the procedure of Toepfer and Lehmann (1961). Based on analyses of the eluate frac- tions with chemical and differential microbiological assays, Storvick et a1. (1964) found that the pyridoxal fraction also contained pyridoxine, and the eluant for pyridoxine contained pyridoxamine. Bain et a1. (1960, 1962) reported a unique method for the separation of the six biologically active forms of vitamin B6 using a single column, containing two resins separated from.each other by a disk of filter paper. The upper layer contained Dowex 50 (K+) and the lower layer Dowex l-formate. When a perchloric acid tissue extract, adjusted to pH 4.25, was applied to the column, the three l6 unphosphorylated forms passed through the Dowex 50 (K+) layer and were bound by the Dowex l-formate. Using a con- tinuous gradient elution method, pyridoxamine phosphate, pyridoxine phosphate, and pyridoxal phosphate, were eluted with a binary system of 0.05 M potassium citrate containing a 0.15 M KCl, pH 4.25 and 0.05 M potassium formate, pH 4.25. Elution of pyridoxal and pyridoxine was accomplished with a solution of 0.05 M potassium citrate and 0.5 M potassium chloride, pH 4.25. Finally, pyridoxamine was eluted with a 0.05 M potassium.citrate and 0.5'M potassimm chloride solu- tion, pH 6.8. Bain's method held considerable promise from the standpoint of separating all forms of vitamin B6. However, S. carlsbergensis assay procedure, which was the best micro- biological method available, was inadequate to identify each component eluted. Chemical Methods Microbiological methods have not proven completely acceptable for the measurement of total vitamin B6 in foods and biological materials. This led to attempts to deve10p a chemical method for the determination of vitamin B6. Colorimetric Following the synthesis of vitamin B6 in 1939, a number of colorimetric methods were devised for the deter- mination of pyridoxine. Most of these methods lacked sensi- tivity, specificity, stability of the color complexes formed, l7 and were only applicable to relatively pure solutions. Recognition of other forms of vitamin B6 also invalidated many of these early colorimetric methods. Spectrophotometric Direct spectrOphotometric determination of pyridoxal, pyridoxamine and pyridoxine were based on their ultraviolet absorption spectra, which showed marked and characteristic perturbations with changes in hydrogen ion concentrations. Melnick et a1. (1945) studied the absorption spectra of pyridoxine, pyridoxal and pyridoxamine in aqueous solutions and reported that each form showed two absorption maxima (254 and 325 nm.for pyridoxine, 251 and 316 nm for pyridoxal and 250 and 325 nm.for pyridoxamine at pH 7.5). Although pyridoxal exhibited a maximum.absorption at 316 nm, its absorption at 325 nm.was approximately the same as that of pyridoxine and pyridoxamine. The ultraviolet absorption spectra of the phosphates of vitamin B6 were reported by Heyl et a1. (1951) and Peterson and Sober (1954). The characteristic absorption spectrum.of pyridoxal phosphate differentiated it from.the other phosphorylated and free forms of vitamin 36' This permitted the quantitative spectrophotometric estimation of pyridoxal phosphate in pure solutions or vitamin B6 mixtures at pH 7.0 by its optical density at 388 nm. An earlier method of Snell (1945) determined pyri- doxamine phosphate spectrophotometrically after its conver- sion to pyridoxal phosphate with a-ketoglutarate. 18 The spectrophotometric method of Snell (1945) was only suit- able for the analysis of a nearly pure solution of vitamin B6. It required that test solutions be free of light-absorbing substances unless adequate blanks were used to correct for their presence. Although no absorption maximum was common to all three components of vitamin B6, the absorption spectra were so close that individual components could not be differentiated. These limitations made the spectro- photometric method unapplicable to the analysis of foods or biological materials. Fluorometry Fluorometry was the most successful analytical tool used for the chemical determination of vitamin 36' Its sensitivity has a distinct advantage over other chemical methods, but until recently its application had been res- tricted to relatively few compounds. Fluorometric assay methods were classified according to whether the fluorescence of vitamin B6 was measured directly or as a chemical derivative. Due to the high sensitivity of spectrophotofluorometry and the problem of interference, applications of the first method were res- tricted to relatively pure solutions. Thus, most of the fluorometric analyses for foods and biological materials were deveIOped for fluorometric derivatives of vitamin B6' Lactone Method Singal and Sydenstricker (1941) discovered that 4- pyridoxic acid, a fluorescence compound found in urine, 19 formed a lactone in the presence of acid which at pH 9 was 25 times more fluorescent than 4-pyridoxic acid. This B6 metabolite was the first of the vitamin B6 analogs to be determined fluorometrically. Fujita et a1. (1955) adapted the 4-pyridoxic acid lactone procedure to the fluorometric estimation of pyridoxal, pyridoxamine, and pyridoxine. The procedure included a method for extraction, hydrolysis, chromatographic separa- tion and measurement of the individual forms of vitamin B6. Quantitative measurement was based on the oxidative conver- sion of the isotels to 4-pyridoxic acid and then to the highly fluorescent lactone form. Pyridoxamine, which could not be oxidized directly, was converted to pyridoxine with nitrous acid, before oxidation to pyridoxic acid. Both Fujita et al. (1955a) and MacArthur and Lehman (1959) reported that the yield of 4-pyridoxic acid lactone derived from.pyridoxamine was comparatively low; MacArthur and Lehman (1959) simplified the lactoniza- tion procedure of Fujita et al. (1955a,b) after separation of the three fomms of vitamin B6 by differential elution from Dowex 50 (Na+). The separation technique yielded a pyridoxamine fraction which was free of pyridoxine, however, this method was not satisfactory for biological extracts. Later Storvick et a1. (1964) found that if one form of vitamin B6 greatly predominated in the extract, the separation of the various components of the vitamin prior to 20 lactonization was not necessary. For example, pyridoxine could be determined in the presence of pyridoxal with minimal interference unless pyridoxal greatly exceeded pyridoxine. This observation eliminated the need to separate the three forms of vitamin B6 provided the vitamin B6 content was pre- dominated by one form of the vitamin and the sample prepara- tion was relatively pure. The lactone method was highly specific and much more sensitive than spectrophotometric or colorimetric methods. However, the existence of interference from.other highly fluorescent materials of biological origin or from food- stuffs has limited the usefulness of this assay. Cyanohydrin Method Bonavita and Scardi (1959a,b) reported that the pro- duct of the reaction between cyanide and pyridoxal-S-phosphate exhibited fluorescent prOperties distinct from.pyridoxal-S- phosphate. A year later Bonavita (1960) discovered that a similar reaction occurred between cyanide and pyridoxal. Subsequently, a method based on this reaction was developed specifically for the determination of pyridoxal and its phosphorylated form. According to the study of Bonavita (1960), pyridoxal- 5'-phosphate cyanohydrin exhibited maximum fluorescence at pH 3.8 with excitation at 315 nm and emission at 420 nm, 'while pyridoxal cyanohydrin exhibited fluorescence with excitation at 358 nm and emission at 430 nm. Studies con- cerning the spectral characteristics of the cyanohydrin '21 drivatives by Bonavita (1960) and Yamada et a1. (1968) confirmed that pyridoxal cyanohydrin and pyridoxal phosphate cyanohydrin exhibited totally different responses with a change in pH. They concluded that more reproducible results were obtained if pyridoxal cyanohydrin was measured at pH 10 and pyridoxal phosphate cyanohydrin was measured at pH 3.5. Pyridoxal or pyridoxal phosphate were also able to be measured in the presence of other components of vitamin B6 because pyridoxamine and pyridoxine and their phosphates lacked the double bound in the C-4 position. This prevented them frmm reacting with cyanide to form.the lactone. MOre- over, pyridoxal phosphate was able to be measured in the presence of pyridoxal unless the latter greatly exceeded the former in concentration. Toepfer and his associates (1960) adapted the method of Bonavita (1960) to the determination of pyridoxal and pyridoxamine. Determination of pyridoxamine required the quantitative conversion of pyridoxamine to pyridoxal via a nonenzymatic transamination reaction with glyoxylic acid (GOA) (Metzler et al., 1954). After conversion to pyridoxal, it was treated according to the procedure of Bonavita (1960). Polansky et a1. (1964) showed that pyridoxine could also be determined fluorometrically by the cyanohydrin method. Pyridoxine was quantitatively oxidized to pyridoxal over a range of 0.001 to 0.5 pg/ml with manganese dioxide and converted to the cyanohydrin form via the procedure of 22 Bonavita (1960). Conversion of pyridoxine to pyridoxal was greater than 90%. Toepfer et a1. (1961, 1964) reported reproducible results with standard solutions of pyridoxal, pyridoxamine and pyridoxine. The fluorescence intensity of the cyano- hydrin derivative was of the same order of magnitude as the lactone of 4-pyridoxic acid. Later, Ohishi and Fukui (1968) and Takanashi (1968) reported that the spectral changes of pyridoxal and pyridoxal phosphate treated with potassium cyanide were not due to the formation of cyano- hydrin derivatives, but the formation of 4-pyridoxic acid lactone and 4-pyridoxic acid phosphate lactone, respectively. These researchers proved that the cyanohydrin form was merely an intermediate in the reaction between pyridoxal and potassium cyanide and in the presence of oxygen was converted to 4—pyridoxic acid lactone. It was the final product, 4-pyridoxic acid lactone, which provided the desired fluoroscent intensity of vitamin B6. The advantages of the fluorometric determination of vitamin B6 were its relative simplicity,sensitivity and specificity for the aldehyde forms of vitamin B6' The use of this procedure for the determination of vitamin B6 in foods and biological materials required the chromatographic separation of the biologically active components of vitamin B6 from the extracts of food and biological materials be- cause of the presence of naturally fluorescing substances 23 which interfered with the determination. Despite the numerous advantages of the cyanide method, very few applications were reported for food and biological materials. Yamada (1970) modified and adapted the cyanide method to the assay of pyridoxal and pyridoxal phosphate in blood and tissue homogenates. Their method was quantitative and reproducible. Takanshi et a1. (1970) used the cyanide method to determine pyridoxal and pyridoxal phosphate content in biological materials. Homogenates of serum, plasma, and tissues were deproteinized with TCA prior to separation and concentration of the vitamin B6 components from.sample extracts with Dowex IX 8 and Amberlite CG 120 columns. The recoveries varied from 93-100% for pyridoxal and 68-90% for pyridoxal phosphate. In 1971 Masukawa and his coworkers modified Takanashi's procedure and included pyridoxamine and pyridox- amine phosphate in their study. Again individual components 'were separated by ion exchange hromatography. Pyridoxamine, pyridoxal and their phosphate forms were determined as 4- pyridoxic acid lactone and pyridoxic acid-S-phosphate, respectively. Recoveries for pyridoxal, pyridoxamine and pyridoxamine phosphate in milk and plasma were 95-104% while pyridoxal phosphate was consistently 90%. l 24 'BIOLOGICAL ASSAYS Procedures were developed to measure the vitamin B6 that was biologically available to animals. One of the earliest biological tests was the cure of rat acrodynia. This was replaced by the rat growth test, in which the vitamin content was determined by growth resulting from the addition of graded doses of vitamin B6 to a diet deficient in that vitamin. Chicken assays were similar but were used less frequently than those employing rats. The method of Sarma et a1. (1947), a modification of the method of Conger and Elvehjem.(l94l), is one of the principle bioassay methods used today. Sarma and his associates developed a basal ration which permitted minimum growth without vitamin B6 and maximum growth with optimum levels of 36' Food or biological materials to be assayed were mixed in the diet, usually at two levelsJand weight gained was recorded for four weeks. Weight gain values were obtained and compared to a standard curve. Sarma et a1. (1947) established that pyridoxal and pyridoxamine incorp- orated in the diet gave slightly lower values than when fed as a supplement with a medicine dropper or injected intra- peritoneally. This presumably accounted for the somewhat lower values which were obtained by bioassay methods when compared to the S. carlsbergensis method. Henderson et a1. (1941) and Sarma et a1. (1947) made use of the rat bioassay method in their estimation of 25 the vitaminB6 content of various food products. Tomarelli et a1. (1955) also employed the rat bioassay to determine the biological availability of vitamin B6 of heated milk products. However, they did not use the bioassay as a means of estimating total vitamin B6 in milk. Tomarelli et a1. (1955) found that the overall effect of heat sterilization on the vitamin B6 of milk resulted in a decrease in bio- logical activity from.one third to one sixth the original content. Assay the same product using S. carlsbergensis showed only fifty percent reduction in biologically avail- ability. This clearly indicated that the bioassay methods generally have lower values than the microbiological assay. Nutritionally, the rat bioassay method was advantag- eous, since its primary concern was to determine the amount of vitamin B6 which was available for use by the animal, rather than the total amount present. However, one must also consider that not all biological systems were identical and results obtained for one species of animal could not be extrapolated to other animals. It was necessary to know how much of the total vitamin B6 consumed was available to the rat, the chick and the human being. The method also had the advantage that no hydrolytic or extractive procedures were required, which simplified the procedure and eliminated a common source of error inherent in the chemical and micro- biological procedures. According to a study by Toepfer et a1. (1963) on the vitamin B6 values of some selected food samples, 26 the bioassay showed 95% confidence limit which were t 25% of the mean value. METHOD Reagents for Chemical and Microbiological Determination 1. Potassium acetate buffers- (a) 0.01 M, pH 4.5- Dissolve 0.981 g KOAc in H20 and adjust pH with concentrated HOAc. Dilute to 1 liter. (b) 0.02 M, pH 5.5- Dissolve 1.96 g KOAc in H20 and adjust pH with concentrate HOAc. Dilute to 1 liter. (c) 0.04 M, pH 6.0- Dissolve 3.92 g KOAc in H20 and adjust pH with concentrated HOAc. Dilute to 1 liter. (d) 0.1 M, pH 7.0- Dissolve 0.815 g KOAc in H20 and adjust pH with concentrated HOAc or 6 N KOH. Dilute to 1 liter. 2. Potassium chloride-phosphate buffer- pH 8.0. in 800 ml H 0 and Dissolve 74.6 g KCl and 17.4 g K HPO 2 4 2 adjust pH with concentrated HOAc. Dilute to 1 liter. 3. Ion exchange resin- Dowex AG 50WX-8 100-200 mesh. 4. Phosphate buffer, 0.4 M, pH 7.5- Dissolve 69.67 g of KZHPO4 in 800 ml H20. Adjust pH with concentrated H3PO4 and dilute to 1 liter. 5. Pyridoxine (PIN), pyridoxal (PAL), and pyridox- amine (PAM) standard solutions. Prepare separate solutions for each as follows: (a) Stock solution-10.0,pgfml. Dissolve 12.16 mg pyridoxine HCl, 12.18 mg pyridoxal HCl, and 14.34 mg pyridoxamine HCl, respectively, in l N HCl and dilute to 1 liter with l N HCl. Store in brown glass bottles 27 28 at 4°C. 6. Potassium cyanide 1.0 M. Dissolve 6.512 g of KCN in H20 and dilute to 100 m1. 7. Sodium carbonate, 0.4 M. Dissolve 4.24 g of Na2003 in H20 and dilute to 200 m1. 8. Glyoxylic Acida, 0.5M. Dissolve 480 mg of GOA in H20 and dilute to 10 m1. 9. Manganese dioxide. Prepared as described by Mancera et a1. (1953). Apparatus for Fluorometric Determination Technicon Fluorometer Model No. IIbwas used for con- tinuous flow analysis. TurnerSpectrophotofluorometerc was used for manual analysis. Samples used for Analysis One food sample was chosen from each of four major food products, namely: dairy (infant formula), meat (ham), cereal (wheat flakes), and legume (lima bean). Commercially processed samples were chosen with the same lot numbers for proper replication on different days of analysis. Two identical samples and recoveries from.each category of food were analyzed during each run and were aSigma Chem. 00., Detroit, Michigan. bTechnicon Instruments Corp., Tarrytown, N.Y. cG. K. Turner Associates, Palo Alto, California. 29 repeated at least three different times. Both chemical and microbiological assays were performed on the same day. Sample Preparation for Fluorometric.Determination of Vitamin B6 Sample extracts and chromatographic separation of the three components of vitamin B6 were carried out as des- cribed in A.0.A.C. (1961, 1970). Sufficient sample was accurately weighed into a 300 m1 Erlenmeyer flask, so that the concentration of total vitamin B6 was 4-10 pg. One hundred and eighty milliliters of 0.44 N HCl was added to samples of plant origin, and 180 m1 of 0.055 N HCl was added to samples of animal origin. Plant products were autoclaved for 2 hours at 121°C. and animal products for 5 hours at 121°C. The samples were cooled to room temperature, adjusted to pH 4.5 with 6 N KOH, quantitatively transferred to 250 ml volumetric flasks and diluted to mark with distilled water. The samples were filtered through a Whatman No. 42 filter paper and a 40-200 m1 aliquot of filtrate was placed on the ion exchange column, which had been previously equilibrated with 0.01 M KOAc buffer (pH 4.5). Pyridoxal (PAL, pyrodixine (PIN) and pyridoxamine (PAMD were eluted from the column with 100 ml portions of 0.04 M KOAc (pH 6.0); 0.1 M KOAc (pH 7.0); and 1.0 M KCl-0.1 M KZHPO4 (pH 8.0), respect- ively. Individual components eluted from the column were treated chemically as described below. 30 Pyridoxal The eluted PAL fraction was diluted with 0.4 M phosphate buffer (pH 7.5) to obtain a final concentration of 0.01-0.05 pg/ml. Four milliliters of the diluted sample were placed in each of two test tubes A and B (B was for blank). One-tenth of a milliliter of 1.0 M.KCN and 0.1 mi of water were added to test tubes A and B, and the tubes were placed in a 50°C water bath for 2 hours. After cool- ing, 2‘ml of 0.4 M Na2C03 were added to each test tube and the fluorescence was measured at 355 nm excitation and 436 nm emission. Pyridoxamine The eluate obtained from the ion-exchange column was diluted with 0.4 M phosphate buffer (pH 7.5) to a final concentration of 0.01-0.05 pg/ml. Four milliliters of the sample were pipetted into each of the two test tubes, A and B (B was for blank). One-tenth milliliter of 0.5 M GOA and 0.1 m1 of water were added to test tubes A and B. The tubes were heated in a 100°C water bath for 15 minutes. After cooling, 0.1 mi of 1.0 M KCN was added to each test tube which were then placed in a 50°C water bath for 2 hours. After cooling, 1.9 m1 of 0.4 M Na2C03 was added to each tube. Fluorescence was measured in the same manner as described .for PAL. Eyridoxine Sample eluate (10-40 ml) was pipetted into a 125 m1 Izlrlenmeyer flask and adjusted to pH 5-6 with 0.1 N HCl. 31 One-tenth gram of M'nO2 was added to each sample and stirred continuously or shaken on a rotary shaker for 30 minutes at room temperature. Sample was centrifuged and the super- natants were filtered through Whatman No. 42 paper into a 100 m1 volumetric flask. Manganese dioxide residue was washed with distilled water, centrifuged and the washing decanted into the original supernatant. The residue was dis- carded. The pH of the supernatant was adjusted to 7.2-7.5 with 0.4 MKZHPO4 and diluted to mark with distilled water. Four milliliters of the diluted solution were pipetted into a test tube and 0.1 ml of 1.0 M KCN was added to each sample. Samples were heated in a 50°C water bath for 2 hours. Fluorescence was determined in the same manner as described for PAL. Blank samples were treated as described above ex- cept that M'nO2 was omitted. Standards Ten milliliters each of lO‘pg/ml standard PAL, PAM, and PIN stock solutions were mixed together and adjusted to pH 4.5 with 6 N KOH and concentrated HOAc. The standard mixture was applied to the ion-exchange column and the three fractions were eluted from the columns and treated as described above. It was found that there was negligible difference between chromatographed and unchromatographed standards. For convenience, therefore, all standards were not chroma- tographed and were prepared directly from.the stock solution. 32 Recovery “S t‘a’n’dards Recovery standards were prepared by adding 1.0 ml of each of the Standard stock solutions of PAL, PAM and PIN (10 ug/ml) to the sample prior to acid hydrolysis. Continuous Flow Analysis For the determination of PAL, 2-8 ml of eluate were pipetted into each of the two test tubes A and B. Suffic- ient 0.4 M phosphate buffer was added to each of the test tubes to obtain a pH of 7.2-7.5 and a final volume of 10 ml. Twenty-five hundredth milliliter of 1.0 M KCN and 0.25 ml of water were added to test tubes A and B respectively, and the tubes were placed in a 50° C water bath for 2 hours. After cooling, the samples were placed in sample cups for continuous flow analysis. The flow diagram of vitamin B6 analysis is shown in Figure 4. A procedure similar to that employed for PAL was followed for the determination of PAM, except samples were treated with 0.25 ml of 0.5 M GOA at 100°C for 15 minutes prior to the addition of KCN. PIN values were determined by the procedure previously described for pyridoxal, except 2.5 m1 of 1.0 M KCN was added to the 100 ml sample prior to the two hour heat treat- ment. The autoanalyzer system was assembled as shown in Figure 4. Water was pumped through all tubes and the base- line adjusted to 5 using a sample aperature of 3 and a 33 reference aperature of 1. After pumping Na2003 through the system.for 20 to 30 minutes, the high standard of one of the three forms of B6 was then used to adjust its mathmn fluorescent response to 95. The sample probe was placed in water and the recorder allowed to return to the baseline be— fore standards and samples were analyzed. Blanks for each sample were determined as described previously. Calculations Each form of vitamin B6 was treated and.measured separately. Fluorescence was proportional to the concentra- tion of the lactone of 4-pyridoxic acid. Blanks were deter- mined for each sample to measure the concentration of non-B6 compounds exhibiting fluorescence. The difference between sample fluorescence and blank fluorescence was used to calculate the vitamin B6 concentration in each sample using the following equation: Fluorescence sample-Blank 100 Fluorescence of l pg’Bg7ml x (Wt. of sampIé’ x dilution factor = ug of PAL or PAM or PIN/g of sample Microbiological Assay for vitamin B6 The procedure for the chromatographic separation and determination of vitamin B6 using S, carlsbergensis was the revised method of Toepfer and Polansky (1970) with the following slight modifications. The innoculum, S. carlsbergensis, were suspended in 500 m1 of sterilized water after the second rinse of the cells. One milliliter of the 34 assay innoculum was then aseptically added to each sample tube using an auto-pipet. Screw caps were used instead of caps with holes. In order to maintain an aerobic condition, free of contamination, caps were loosely fitted on to the test tubes. Samples were incubated at 30°C in a NBS Gyrotory at a speed of 250 RPM. RESULTS Extraction Acid hydrolysis and enzymatic hydrolysis of vitamin B6 in various food products were examined in this study. Samples treated with 0.1 g each of Taka-diastase and papain for 4-6 hours at 37°C prior to acid hydrolysis were found to have total vitamin B6 values higher than those samples receiving only acid hydrolysis. However, analysis of the enzymes indicated the presence of vitamin B6 in these enzymes (Table I) . The acid hydrolysis described by Toepfer et a1. (1961) was adapted for all samples studied. No significant differences were observed between the samples with recovery standards added prior to autoclaving and those with recovery standards added after autoclaving. Therefore, the recovery standards were added prior to autoclaving for all the samples reported in this study. Separation of Pyridoxal, Pyridoxamine, and Pyridoxine by COlumn Chromatography Dowex AG 50W-X8 was used for separation of PAL, PAM and PIN from.food extracts (Toepfer et al., 1961). After elution each form was chemically and microbiologically analyzed. 35 36 Mixture of PAL, PAM; and PIN standards were combined and treated similarly to the sample. Chemical determination of the standards after separation on ion-exchange columns indicated that the columns provided nearly perfect separation of the three forms of the vitamin. Recovery of PAL, PAM and PIN from the column were 102.5%, 95.0% and 104.1%, respectively. Fluorometric Determination of PAL, PAM and PIN Standards Standard curves for PAL, PAM and PIN using the fluorometric method are shown in Figure 1. Fluorescence was linearly related to the concentration of PAL, PAM and PIN over a range of 0.01-0.08 pg/ml of solution. Micrdbiological Determination of PAL, PAM and PIN Standards Standards curves of PAL, PAM and PIN were deter- mined from.the relative growth of S. carlsbergensisx and normally varied from analysis to analysis. Freshly pre- pared standards were, therefore, analyzed along with each sample. Figure 2 shows representative standard curves for the microbiological determination of PAL, PAM, and PIN in ham. The concentrations of standard PAL, PAM and PIN (ng/ml) were plotted against percent transmittance (%T) which was obtained from an average of triplicate readings. An analysis of the slopes showed that PAM exhibited the least sensitivity with a slope of 0.074. PAL was only slightly more sensitive than PAM with a slope of 0.075, while PIN exhibited the greatest sensitivity with a slope of 0.089. 37 Vitamin B6 Contents in Selected Food Products The content of pyridoxal, pyridoxamine, and pyridoxine determined in infant formula, ham, cereal, and frozen and canned lima beans using both microbiological and chemical methods are shown in Tables 11a and b, 111a and b, IVa and b. Pyridoxal Data in Table 11a and b represents the PAL contents measured in various food products. Chemically determined PAL values were approximately 3-12 times higher than those measured using the microbiological method. Recoveries determined by the fluorometric method varied between 83 -97% with a standard deviation ranging from 3-13 for the different food products. Recoveries for the microbiological method varied from 53-106% with a standard deviation ranging from 7-19. Values corrected for percent recovery showed a slightly lower standard deviation than the uncorrected values. Pyridoxamine Data in Table IIIa and b showed the pyridoxamine con- tent in various food products. PAM values obtained by the chemical method were approximately 2-3 times greater than those obtained with the microbiological assay. With the exception of ham, the percent recoveries for most of the samples using the chemical method varied from 86 -1111 with a standard deviation ranging from 2 -11 depending on the product. Ham had a constant recovery of approximately 66%. 38 The percent recoveries using the microbiological method exhibited a range of 74-1131 with the standard deviation varying from 7-23. Values corrected for percent recovery exhibited a slightly lower standard deviation for most of the food samples analyzed. Pyridoxine The PIN values of various food products are shown in Table IVa and b, Ham and frozen lima beans had only a small amount of PIN, while canned lima beans contained a negligible amount of PIN in the sample size used for analysis. High PIN values were found in the cereal and infant formula. Values obtained from the chemical determination of PIN were 1-2 times as great as those values obtained from the microbiological method. The percent recovery for the various food products analyzed using the chemical method varied from.55-74' with a standard deviation ranging from 3-7. The percent recovery from the microbiological method varied from.99-l32 with a standard deviation ranging from 8-31. Comparisons of Fluorometric and Microbiological Total Vitamin 36 Values Table V shows the comparisons of total vitamin B6 values for various food products obtained by the micro- biological and chemical methods. The comparison also includes published values which were determined using the 39 S. carlsbergensis assay organisms. Values obtained by the chemical method exhibited almost twice as much total vitamin B6 as those obtained by the microbiological method. However, the values for lima beans determined by the chemi- cal method were 5 times greater than when determined micro- biologically. A bar graph representation for various food pro- ducts comparing the fluorescent response of PAL and its blanks is shown in Figure 3. Sample Blank Study Table V1 is a study of Vitamin B6 standards and sample blanks which have been treated with ultraviolet light or [(111104 prior to the addition of KCN. Since ultraviolet light and 1041104 reacted with substances other than PAL, PAM and PIN in the food extract, thus, no conclusions could be made from this study. Another study was conducted in this laboratory to determine the interference of thiamin in the fluorometric determination of vitamin B6. Results indicated that KCN caused thiamin to partially fluoresce at the same wave- length of pyridoxal. However, when thiamin and pyridoxal were chromatographed over a Dowex AG 50W- X8 column, thiamin was not detected in the pyridoxal eluate. This would indi- cate that thiamin was not an interfering substance in the fluorometric determination of pyridoxal provided the sample extract was passed over the ion exchange coltmm. Thiamin 40 interference in determination of PAM or PIN was not of concern because sample blanks would account for any fluores- cence due to thiachrome formation. 41 .oahuao w~.\om w1.m« ofihufio emu mo coauouuaoosoo asap .cameom mo we 00H one unnummwonmxoa mo wa_ooH ponamunoo mxcwan «Shanna xsdan oahnao one NH.o H~.o mo.o Aaoueaouasa seesaw a vague mamawm coosuon mucouomman nm~.o ama.o BHH.o seamen seesaw am.o am.o aa.o Aaaueaouaea «ahead w vaouvoagamm aa.o ma.o oe.o AaousHonaaa anode «Hagan zHa sad Sam ucoaumoua Aw\wav zoaaamazmozoo .manahom ucomnw aw om nfiauufi> mo coauocaauouoo man you mwmhaouomn owed one oaumahnco mo GOmwumaaoo < .H mum¢H 42 H.~ n.H 00.0 0H.0 n 0 «0.0 HH.0 .>o0.oamum 0.0 H.0 HH.0 00.0 «0H n0 NH.0 00.0 com: «.0 0.0 0H.0 00.0 00 e0 0H.0 o¢.0 d 0.0 0.0 HH.0 H0.0 00 00 HH.0 00.0 0 0.n 0.o 00.0 no.0 0HH n0 0H.0 no.0 N 0.n 0.o 00.0 0o.0 0HH n0 0H.0 no.0 H Bum n.0 0.0 «0.0 00.0 N a 00.0 n0.0 .>oa.ocwum n.0 0.o H¢.0 d0.H 00 00 «0.0 0N.H com: «.0 0.o 00.0 n¢.H an 00 00.0 00.H N 0.0 «.0 00.0 oo.H Ho 00 00.0 NN.H H mamwmummww «.0 H.0 H0.0 no.0 o 0.0H 0.0 NH.0 .>on.ocou0 0.0 0.0 00.0 0¢.H mHH 00 00.0 N¢.H can: H.¢ 0.0 00.0 0¢.H 0HH n0H 00.0 00.H N n.0 0.0 00.0 00.H 0HH 00 00.0 00.H H acmwmowwww .uuou .Huoocb ouon. .6050 oHon. .aono ouon. .5050 * oHaamm uosoonm ouon\aonu mo oHumm oaHm> .Huoo nuo>ooom N osHu>.Hoaxm oHQEMm mo w\0n. .moonuoa.HononoHooHOHa_oco oHuuoaonoaHm emu no oonaahouoo mngawm ooom wouooHom cH moaHm> HoxooHHnm .mHH MH0on .0cuum n.0 H.o No.0 0H.0 0o 00 H0.0 00.0 new: H.¢ n.n No.0 00.0 n0 00 H0.0 00.0 a 0.0 o.o No.0 HH.0 n0 o0 H0.0 00.0 0 0.0 0.0 No.0 00.0 H0 o0 No.0 no.0 N n.0 o.¢ No.0 0H.0 H0 Hn No.0 no.0 H oHnahom unomaH H.H n.0 00.0 00.0 0H HH 00.0 nN.0 .>o0.ocou0 0.HH 0.0H n0.0 00.0 o0H n0 n0.0 00.0 new: 0.0H 0.0H 00.0 nN.0 00 n0 00.0 no.0 q 0.0H N.0H N¢.0 0N.0 00 00 n0.0 0n.0 0 o.NH 0.HH 0N.0 Ho.0 «NH NHH o0.0 o0.0 N o.HH 0.0 00.0 00.0 0HH n0 H0.0 H0.0 H Hmouoo .Hnooca .uuoo .oHOHz .Bonu .oHon cause .oHOHz eaono * oHaamm uodooum oHOHznaoso mo oHumm oaHm> .uuoo nno>ooom N osHm>.noaxm oHQEMm mo 0\wa. .moonuoa HmuHmoHoHnouoHE one oHHuosouoaHm may no oocflauouoo moHeaom ooom nouooHom aH mosHo> meooHnnm .oHH mqmma .oouum N.N o.N mn.N 00.0 on oo o0.H no.0 ado: H.N n.H no.N 00.0 00 no HN.N 0n.0 o 0.N n.H H0.N 00.0 H0 oo 0n.H no.0 0 0.H H.N H0.N No.0 Ho no 0n.H no.0 N «N o,.N oN.N No.0 Ho no 00.H no.0 H Bum m.N 0.o No.0 0H.0 HH HH 0.0 no.0 A>oo .oamum a.NH 0.NH 0H.0 00.N 00H HHH 0H.0 00.N can: o.oH 0.NH 0H.0 o0.H n0 0HH 0H.0 N0.N N 33533 N.¢H 0.NH oH.o nN.N 0HH 00H 0H.0 0N.N H meson daHH H.o o.N H0.0 00.0 n n0 no.0 00.0 .>o0 .osdum n.n 0.o 0N.0 00.H HHH «NH oN.0 mo.H ado: o.m 0.o 0N.0 00.H oHH 00 0N.0 00.H N 8388 n.n H.0 0N.0 00.H ooH 00H NN.0 00.N H mason daHH .Huoocs .uuou .0.»on .35 .3on .628 .0.»on .598 a» «H.930 Undone 9805328 no 3530 oaHo> .unoo nuo>ooo0 .n oaHm> .uoexm 308mm mo 0 \01 .3939: HoonoHoHoouoHa one oHuuoBouonHm 93 no oocHauouoo moHnHaom ooom oouooHom cH moaHSr osHamxooHHnm . UHHH flfih q CEGO CNCI' .mpoauofi HmonoHoNQOHUNB new ONHUUBOHODHN USU kn DOCHENOUQU QOHQEQQ DOOM. DUUOOHQQ EN. QQDHW.) UCHENKOUHMKAAN .QHHH NNNNN «.0 H.0 H0. H0.0 0N N H0. 00.0 .>o0 .oaoum o.N n.H 00. HN.0 0HH 00 HH. 0H.0 new: .n.N 0.H 00. 0N.0 nNH 00 0H. 0H.0 a 0.N 0.H 0H. 0N.0 nNH 00 0H. 0H.0 0 0.N n.H 00. HN.0 NNH o0 HH. 0H.0 N 0.N 0.H n0. HN.0 0n 00 0H. 0H.0 H oHsauom unumcH .0 0.0 0.0 00.0 No.0 0H o 00.0 0N.0 .>o0 .ocmum o.H 0.N o0.N no.0 00H 00H 0¢.N on.¢ coo: 0.N 0.N 0H.N no.0 00H HOH oN.N 00.0 a 0.N 0.N oN.N 00.0 00 HOH 00.H 00.0 0 0.H 0.H 0n.0 N0.o on HOH 0n.N N0.o N N.H 0.H mn.0 00.0 Nn 0HH Hn.N o0.0 H Huouoo .Huoo .Huooaa .ouon .Eooo .ouonfl .aooo .ouon .aooo % oHaaww oodooum ouodanuooo mo 0.3mm osHo> .uuoo oHeamano>muMwo .n 0.H—Ho.» .Hoexm .mooouoa_Hon0oHoHooH0Ha one oHuuoaouosHm oou no oocHahouoo moHaamn ooom oouooHom eH moon> oaHawxooHunm .oHHH 505 46 0.0 H.o 00.0 00.0 H0 0 n0.0 00.0 .>oo .ocmum 0.H 0.0 00.0 0N.0 00H 00 0H.0 nH.0 sums enhances un nn nu N0 Hm oo poncoo a venomous nn nn nu 00 H0 oo noncoo 0 o.H n.0 «n.0 0N.0 0NH 0m NN.0 nH.0 N 0.N 0.0 0H.0 0N.0 HaH 0n 0N.0 nH.0 H _am: nu nu nu nn 0H 0 un uu .>oo .oawum nn nn un nu 00H 0n nn nu coo: venomous nu nn nu nn n0H Hn oo voodoo N oouammoa Aaououmv nu nn un nn 0NH on oo uoanoo H modem maHH 0.0 H.o 00.0 00.0 0 00.0 H0.0 00.0 .>00 .oooum 0.H n.0 0N.0 0N.0 NOH 00 00.0 nH.0 coo: N.H o.0 0N.0 0N.0 n0H 00 N0.0 nH.0 N . 83:30 0.H 0.0 0N.0 0N.0 oNH 00 0N.0 nH.0 H mamom eaHH .Huoo .Huooca .oHon saoou .onon .aooo .ouon_ .aoou * onaum uonooum oHonnaoou mo OHumm ous> .Huoo nuo>ooom N onHo> .Hoexm oHnHBmm mo 301 .mooouoa.HoonoHoHoouoHa 0cm oHuuoaouosHm oou no oocHauouoo moHeamo ooom oouooHom aH mosHm> oconoHHnm .m>H 0H0oo .ocuum 0.H N.H NN.0 H0.0 00 on 0N.0 NN.0 coo: 0.H 0.H nH.0 0N.0 nNH 0n 0H.0 0H.0 o 0.0 0.H 0N.0 oN.0 0n N0 0H.0 0N.0 0 0.H 0.H oN.0 00.0 nn 0o 0N.0 0N.0 N 0.H H.0 0N.0 n0.0 0HH 0n oN.0 oN.0 H H.0 00.0 NH.NH 00.HH nH n 00.0 oN.0 .>oo .0cmum N.H 0.0 Nm.0N Nn.00 00H Nn oH.0N N0.¢N coo: H.H 0.0 00.00 NH.no «0 no N0.n0 NN.N0 o 0.H 0.0 00.00 H0.00 NNH 00 no.o0 0o.H0 0 0.H 0.0 «0.0H no.0N NHH 0n 00.HN 0H.nH N H 0.H 0.0 00.0H no.0N NHH 0n o0.HN 0H.nH H Hoouoo .Huou .uuooco .ouon eaooo .ouon .aoou .OHOHSH .amoo % «Hmaom nonooum ouonnaooo mo OHumm 05Ho> .Huoo num>ooom N oaHm> .Homxm ngamm mo .aw\ma .mooouoa HmonoHOHooHOHB 0am 0HuuoaouosHm oou no oooHauouoo moHaawm ooom oouooHom GH mouHm> oaonoHunm .o>H mH0 .amno soon .uwa .n ..3 u .uamaaoa was NemaaHoao .HomH .on .m gauge econ .uma .n ..a .ouaaaaoa was m .manaaao ..m.m .xoooa «N.H NN. Hm. mo. HN. No. oH. Nm. Ho. anm. .Haauoa nadoaH mm.H Nn.0N Nn.mm om.N na.a nm. mo.a mo.Hm NN.Na oo.mN Haauoo mo.~ ma. ow. mm.N oa.m HH. mm. mo.m am.o an.m .aam oo.a mN. aN. HNH. mo.N oa. ma.H om. no.m am.H Aaououmomaaum Na.a nn nn omN. mm.H ,mm. ma.H on. oN.N «H.H Homaaaooaaaa ouon\.Eooo .ouoHS .895 6.3.30 .895 .ouon .aoou .oHon .Booo Houon. uofiooum oHa 0 canon? Houou mo cooHuonHa—oo .> ".3040. 49 .Hsoo ”macs .wnwum nn 0.0 0.HH 0.0 nu 0H. 0.0 nu 0.H .umouu oczm .. o.HH o.om 0.o .. o.oH o.n .. o.OH .uaoup «case .330 0.0 un 0.0H 0.0 nn 0.0 0.0 nn 0.H .umouu .>.= 0.o nu 0.oN 0.H nn 0.HH 0.o uu 0.n .umonu .>.D 0.0 nn 0.H 0.H 0.0 0.0 0.0 nn 0.NN .onmum 0.o nu 0.0 0.0 0.0 0.n n.0 uu 0.0H xcmHm 0.0 un 0N 0.H 0.0 0N 0.0 nu N.N .ocwum 0.o nn 00 0.0 0.0 0.0N n.n nu 0.0H oHnawm zu o\3 zu 0\3. zo\3. zo 0n3 zo 0\3_ zo\3 zo 0\3_ 20 0x3. zo\3. udoauooua 080 o\3 090 050 0\3 090 090 0\3 emao sz Edm H mHmoH o s zc_ea .mzaxonaa>a .aa «on m>m=u am 0520mean «on. zmn0>m 0~0>4a