Talanta 51 (2000) 799 – 806 www.elsevier.
Simultaneous determination of water-soluble vitamins excreted in human urine after eating an overdose of vitamin pills by a HPLC method coupled with a solid phase extraction
Chan Mo Cho, Joung Ho Ko, Won Jo Cheong *
Department of Chemistry and Center for Chemical Dynamics, Inha Uni6ersity, Incheon 402 -751, South Korea Received 22 February 1999; received in revised form 29 September 1999; accepted 10 December 1999
Abstract We have applied a quick and convenient method for determining water-soluble vitamins excreted in human urine. We found that the Sep-Pak C18 cartridge was useful for preconcentration and recovery of water-soluble vitamins in urine with minimized loss of vitamins. The recovery of vitamins was well over 90%. The separation was carried out by gradient elution with 90/10 (v/v%) methanol/water with 0.1% triﬂuoroacetic acid (TFA) and water with 0.1% TFA on a mBondapak C18 column. The separation was completed within 15 min. We measured concentrations of water-soluble vitamins excreted in urine after swallowing an overdose of vitamin pills on purpose, and found that the concentration of each vitamin increased rapidly to the maximum in 2 – 3 h and decreased swiftly. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Water soluble vitamins; HPLC; Solid phase extraction
1. Introduction Lack of some nutrients causes serious diseases for humans even though small amounts of them are required to maintain good health [1 – 3]. The dispensable nutrients are called vitamins and are supplemented by eating appropriate food. They can be categorized into two groups, water-soluble and fat-soluble vitamins. Many analytical meth* Corresponding author. Fax: +82-32-8675604. E-mail address: email@example.com (W.J. Cheong)
ods for qualitative and quantitative determination of vitamins have been developed. A relatively small amount of studies on water-soluble vitamins excreted in urine have been done. High performance liquid chromatography (HPLC) techniques show some merits in view of simplicity and speed [4–6]. Rapid analysis is helpful since vitamins are easily broken by light, and are unstable in urine. Solid phase extraction is necessary prior to HPLC to remove interfering components. Analyses of vitamins in commercial vitamin pills are much easier since they contain much higher level of
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beverages. Switzerland). B6. (4) vitamin C. (3) vitamin B6. however. but individually identiﬁed . nicotinamide and nicotinic acid extracted from various foods. causes some anomalies. Louis. USA). Recent studies for quantitative HPLC determination of water-soluble vitamins are as follows: Powers et al. and B6 were obtained from Aldrich (Milwaukee. Moreover inorganic buffers are not compatible with mass spectrometry and should be avoided if one considers LC/MS (liquid chromatography/mass spectrometry) applications. Inorganic buffers or ion pairing reagents were added to the eluents in previous studies.15]. and pharmaceutical tablets [14. [12.M. (2) vitamin B2. and 5-methylcytosine (as an internal standard) from Sigma (St. Thorough cleaning with pure water should be done ﬁrst. The structures of vitamins and internal standard. Experimental
2. Each component was determined separately by a particular HPLC method.  simultaneously determined vitamin B1. we have developed a convenient HPLC method coupled with a solid phase extraction for determination of water soluble vitamins in urine without using an inorganic buffer.  reported extraction and quantiﬁcation of vitamin B1. 2. Papadoyannis et al. and in most of the relevant studies. The capillary zone electrophoresis techniques is also a powerful tool to which a lot of interest is focused in determination of vitamins in foods. vitamins were not simultaneously determined. Reports on determination of water soluble vitamins in urine were relatively limited. B2. Inorganic salts can build up in the ﬂow line elements such as check-in and check-out valves to result in malfunction of check valves. effervescent tablets. and folic acid in tablets by gradient elution with water and methanol including an ion pairing reagent. Switching from an aqueous buffer to an eluent of high content of organic solvent should not be done directly. Agostini et al. drugs and biological tissues or ﬂuids [4 – 6]. Cho et al. The HPLC grade
Fig. beverages. (1) Vitamin B1. Blanco et al. (5) internal standard (5-methyl cytosine).
.  determined nine water and fat soluble vitamins in pharmaceutical preparations and vitamin-spiked blood and urine samples by solid phase extraction followed by dual HPLC analyses. B2 and B6 from patients. There has been a large number of HPLC studies on determination of vitamins in foods. USA) and used without further puriﬁcation. B2. nicotinic acid. executed similar research . In this study.  studied simultaneous determination of vitamin B1. 1. nicotinamide. from Fluka (Buchs. Use of inorganic buffers. / Talanta 51 (2000) 799–806
vitamins than food stuffs .13]. etc. 5. Vitamins B2. The urine samples were taken 1. B6.1. Vitamins excreted in urine have not been simultaneously determined by a single HPLC run so far although simultaneous determination of the vitamins-spiked urine sample  was recently reported. and 8 h after eating an overdose of vitamin pills. Chemicals
The structures of the internal standard and vitamins are given in Fig. 1. Ion pair chromatographic tech-
niques have been continuously used in determination of water-soluble vitamins in foods. Gennaro et al.
C. 3. vitamin B1 and C.
We used Chromate 3. Rheodyne (Cotati. The sample was eluted with 1 ml water (pH 4.C. Cho et al. and changed linearly to 10% A+ 90% B in the next 7 min. Japan) LC-10AD pumps. The solid phase extraction scheme is summarized in Scheme 1. The solid phase extraction procedure. Apparatus and chromatographic conditions
The HPLC system was composed of two Shimadzu (Tokyo. 5m. I. B6 10 mg. USA) was used for separation at room temperature. / Talanta 51 (2000) 799–806
ent composition was initially 99. An Orion (Beverly. A mBondapak C18 column (250×4. B2 2.5. Milford. We used the Sep-Pak C18(500 mg) catridges to remove most of the interfering components.005 M HCl solution drop by drop with stirring until its pH reaches the predetermined value.1 mg. Preparation of standard mixture
Vitamin C 4.18]).
. The recovery rate was calculated by the following equation. The elu-
2. Stability of vitamins is known to be improved by addition of HCl ([5. The mixture was stored in a dark and cool space (below 4°C in a refrigerator). held for 5 min.M.6 mm.5% B.3.
2. SPE (solid phase extraction)
Urine consists of many components that cause chromatographic interferences with vitamins.5% A+ 0. Korea) SLC 2000 UV-VIS detector. The raw urine was taken from a healthy man in the early morning before breakfast and examined to make sure there was no vitamin detected. we simply compared the area of each vitamin peak in a SPE treated spiked urine and a diluted standard. USA) 7152 injector with a home-made 100 ml sample loop.17.2 mg. and a Samsung (Seoul.1% TFA containing water (A) and 0. Korea) and were ﬁltered and degassed for 30 min before use. Separation of vitamins was carried out by gradient elution with 0. we then loaded an 1 ml urine sample.2 mg. Korea) to process the raw chromatographic data. The acidiﬁed water was prepared by adding a 0.7).2. The sample was ultrasonicated for 10 min before injection.1% TFA containing 90/10 (v/v%) methanol/water (B). The ﬂow rate was 1 ml min − 1.S. First. The diluted standard was prepared by mixing the same volume of the standard mixture and water.0 version software made by Youlin-Gisul (Sungnam.
Scheme 1. (5-methylcytosine) 7. we ﬂushed the stationary phase with 2 ml methanol and 2 ml water adjusted at pH 4. USA) model 520 digital pH meter was used to adjust pH of sample solutions.
2. The detector wavelength was set at 254 nm.9 mg were measured and dissolved in 50 ml water including 0.2) followed by 2 ml methanol at a ﬂow rate of 1 ml min − 1.
methanol and water were purchased from Duksan (Seoul. The spiked urine was made up of the same volume of the standard mixture and the raw urine (50:50). Reco6ery test
In order to measure the loss during the SPE procedure.4. B1 8. The eluents were collected in a bottle and evaporated to dryness.1% (v/v) 1M HCl. The residue was dissolved in acidiﬁed water (pH 3.2 to activate the stationary phase.
Results and discussion Separation of vitamins was accomplished within 15 min in our method. A mBondapak C18 column (250 × 4. the area count of the vitamin component. and 10 mg B6. the response factor ratios were computed. the pre-determined response factor ratio. (5) vitamin B2. The eluent composition was initially 99.1% TFA containing 90/10 (v/v%) methanol/water (B). Watersoluble vitamin are known to be almost excreted in 2 8 h after dosage [1. Collection and analysis of urine after dosage of 6itamins
We observed the level of each vitamin in the urine of a volunteer after his swallowing an overdose of vitamins (three pills). (4) vitamin B1. Cho et al. 2. Next.X. 5. and RR. 5m) was used for separation at room temperature.802
C.2].6. 10 mg B2. and 8 h after eating the vitamin pills. The response factor ratio (RR) is deﬁned as the response factor of a vitamin component divided by the response factor of internal standard. and C.M. / Talanta 51 (2000) 799–806
% recovery =100 ×(peak area of each vitamin in the SPE treated spiked urine)/(peak area of each vitamin in the diluted standard mixture) Three standards of different solute concentra-
tions were used.5% A + 0. 3. The response factor (Rf) for individual components were determined from the data of the standard solution as follows: Rf = M/C In the above equation.
Fig. Then a urine sample was analyzed and the concentration of the vitamin component (Cx) in the urine sample was calculated as follows: Cx = CIS(Mx/MIS)/RR.6 mm. Then the supernatant was taken and 0. The vitamin pill was composed of 70 mg vitamin C. The chromatogram of a standard mixture is shown in Fig. 2. The results of recovery test for the solid phase extraction are assembled in Table 1.X where MIS is the area count of internal standard in the urine sample. The ﬂow rate was 1 ml min − 1. The standard solution was ﬁrst analyzed. The chromatogram of standard mixture of water soluble vitamins. M denotes the measured area count for a component. Mx. 2. The detector wavelength was set at 254 nm.
3. held for 5 min. 50 mg B1. and four replicate experiments were carried out for each standard.5% B. We collected urine samples 1. Each urine sample was stored in a refrigerator under 4°C and was centrifuged at 10 000 rpm for 20 min.
2. Separation of vitamins was carried out by gradient elution with 0. (3) vitamin B6. (1) Vitamin C. The recovery rates
. (2) internal standard.1% TFA containing water (A) and 0.1%(v/v) 1M HCl and the internal standard were added. and changed linearly to 10% A+ 90% B in next 7 min. its concentration.
and 3. and water.3) (3. 3 was collected much later. B6. we can note that retention times of vitamins in Fig.24×10−4 1.7 mg ml − 1 (5. 2.80×10−5 1.M. We were unable to use different wavelengths for individual components during a chromatographic run since the detector we used was without timed-program capability. Vitamins taken at overdose proved to rapidly show up in the urine. 2-propanol. It was usually less than 20% of the swallowed amount. 0. B2. Cho et al.1 99.16×10−5 9.6) (5. etc.3× 10 − 5 M) for vitamins B1. C. B2. were 1.42. Accumulation of such polar species happened actually. Vitamins are quickly metabolized when they exist in excess in a body. 2. and B6.3× 10 − 6.4 95.59×10−4 4. although the chromatogram for a SPE treated urine taken after eating an overdose of vitamins appeared as if there were only the components we searched for (Fig. Such trends were observed for a healthy man (Table 2).9 92. and monitoring the concentration of an interested component in urine will be helpful for clinical purposes. Therefore.4 (1. Vitamin B6 was not detected in its original form in the urine even at 280 nm. The chromatogram of a SPE treated urine collected 3 h after eating three vitamin pills is shown in Fig. B2. a SPE treated urine sample might include non-UVabsorbing components from urine such as saccharides.16×10−4 9. and executed an on-time calibration avoiding errors from variation of response factors.9 93. Comparing Fig. 2 and Fig.
.87×10−5 3. anyway. the signals for other vitamins were dramatically decreased. amino acids.76×10−4 1. It should be noted that the total amount of each vitamin excreted in its original form in urine after eating an excessive amount of vitamins was far less than the amount swallowed. When the optimum wavelength (280 nm) for vitamin B6 was used.8 99. 4).3 94. peak shapes.4) (5. 3. 3 as an example. and peptides.80. Thus we decided to measure chromatographic peaks at 254 nm.C. respectively. The detection limits.2 97. the concentration of a component in urine should be a measure of its present level in the body.0 93. The chromatogram in Fig.76×10−5 97. but far off from that of vitamin B6.8) (3.1)
B1 B2 B6 C B1 B2 B6 C B1 B2 B6 C
Standard deviations are in parentheses.60×10−4 3. The level of other vitamins tended to increase rather sharply to the maximum in 2–3 h and decrease swiftly. and 6. / Talanta 51 (2000) 799–806 Table 1 Recovery rates of water-soluble vitamins in spiked urine by solid phase extraction at three different solute concentrations based on comparison of peak areas between the extract and the standarda. and C and to remove other UV-absorbing species.8) (6.6 90. Such UV-transparent contaminants might accumulate in the column and alter retention times of the vitamin components.6) (4.b Standard c 1 Vitamin Concentration Recovery (%) (mol l−1) 4.6 90.2) (9. We successfully made the column free of contaminants later by a long exhaustive multistage cleaning procedure using acetic acid-added acetonitrile.2) (3. and consequently detector response factors. Such recovery yields are comparable to the recovery rates that Papadoyannis et al.6) (8.80×10−4 1. However. and C. 3 were longer than those in Fig.9) (3. Vitamin B6 seemed to be rapidly metabolized or changed to other species in the body. Nevertheless.8× 10 − 7. 5. Vitamin B6 was not found in the urine samples probably owing to its rapid metabolism in the body. It should be noted that our SPE method was developed primarily to maximize recoveries of vitamin B1. Similar trends were observed for another man even though some differences in excretion rates between two persons were noted (Fig.5 94.72×10−5 4. measured by sequential dilution of the standards of individual vitamins at 254 nm and with a S/N ratio being 5.
are well over 90% in general. we always analyzed a fresh standard mixture right before the SPE treated urine sample. obtained for vitamins-spiked urine samples . 0.4× 10 − 6.22.72×10−4 4. The detector wavelength (254 nm) was close to the optimum value for vitamins B1. 3). 2 was obtained in the early
stage of this study while the chromatogram in Fig.5) (2. Four replicate measurements for each concentration.
Fig. (2) internal standard. The chromatogram of water soluble vitamins in the urine sample taken 3 h after eating vitamin pills. (4) vitamin B2. Conclusion We have applied a method of determination of water soluble vitamins in urine by coupling the
solid phase extraction to gradient chromatographic separation without using an inorganic buffer to simultaneously determine vitamins in urine after eating an overdose of vitamin pills. (1)Vitamin C.804
C. Cho et al.M. 3. (3) vitamin B1. / Talanta 51 (2000) 799–806
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Fig. Min-Eum Press. (C) vitamin C. H.K. K. H.J. / Talanta 51 (2000) 799–806 Table 2 The determined concentration of water-soluble vitamins excreted in urine based on the internal standard method Hour 1 Vitamin C B1 B2 C B1 B2 C B1 B2 C B1 B2 C B1 B2 Area (mV) 86 423 Not detected 19 5074 93 999 290 877 170 496 210 924 461 589 216 690 61 040 305 264 192 044 9631 57 016 113 760 Concentration (M) 1. Parent. Agostini.41×10−5 2.A. J. Macrae (Ed.
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