Determination Of Retinol Research Articles

HPLC method for simultaneous determination of retinol, α-tocopherol and coenzyme Q 10 in human plasma

A simple HPLC method with UV detection is proposed for the simultaneous determination of three lipophilic vitamins: all- trans-retinol, α-tocopherol and coenzyme Q 10 (ubiquinone) in human plasma. The following chromatographic conditions were used: RP-18 column, a mobile phase consisted of methanol – n-hexane 72:28 (v/v) and UV detector set at 324, 292 and 276 nm for all- trans-retinol, α-tocopherol and coenzyme Q 10, respectively. The linearity range was 0.35–70 μM for all- trans-retinol, 0.23–44 μM for α-tocopherol and 0.12–23 μM for coenzyme Q 10. Deproteinised plasma samples were extracted with n-hexane prior to the analysis. The within-day and between day reproducibilities were 1.5 and 3.7% for all- trans-retinol, 4.0 and 5.8% for α-tocopherol and 2.3 and 3.1% for coenzyme Q 10, respectively. Using the proposed method the following recoveries were achieved: 91% for all- trans-retinol, 86% for α-tocopherol and 88% for coenzyme Q 10. The method was applied to the determination of the levels of retinol, tocopherol and coenzyme Q 10 in plasma of healthy children and children treated by elimination diet.

Automated method for the determination of fat-soluble vitamins in serum

A new fully automated high-performance liquid chromatography (HPLC) method using 1 ml of serum has been developed for the determination of retinol (Vitamin A), α-tocopherol (Vitamin E), 25-hydroxyvitamin D 3 and 24 R,25-hydroxyvitamin D 3. The eluate was monitored with a photodiode-array detector at three wavelengths—namely: 265 nm for Vitamin D 3, 291 nm for Vitamin E and 325 nm for Vitamin A. The detection limits were equal to or lower than 1 ng ml −1 for all vitamins. The linearity obtained with serum samples (standard addition method) gives correlation coefficients ( r 2) ranging between 0.999 and 0.996 in all cases, with standard deviation of the slope between 3.2 and 1.6%. The repeatability was between 4.0 and 6.0% and the within-laboratory reproducibility was lower than 10% in all cases. The most outstanding features of the present method are its ease of use, its rapidity and fully automation, which enables its use for routine analysis. The time required per sample was 30 min, because the overlapped development of the steps. This method was used for the determination of normality range of these vitamins in healthy people in the 18–80-year-old interval.

Application of linear discriminant analysis to the biochemical and haematological differentiation of opiate addicts from healthy subjects: a case-control study.

Biochemical and haematological parameters of nutritional interest were determined in the serum of opiate addicts in order to compare them with those obtained in healthy subjects. Linear discriminant analysis was applied for the differentiation of the opiate addicts. Sera of 106 opiate addicts in detoxification treatment (n=19) or in Methadone Maintenance Treatment Program (MMTP) (n=87) were studied. : The determination of classical biochemical and haematological parameters in blood samples was carried out using standardized methods. Determination of retinol and alpha-tocopherol was carried out by high-performance liquid chromatography with diode-array detector. Folic acid and vitamin B(12) were determined using competitive binding techniques. Minerals were determined by flame emission spectrometry (Na and K) and atomic absorption spectrometry with air-acetylene flame (Ca, Mg, Fe, Cu and Zn) or with hydride generation (Se). Phosphorous was determined using a colorimetric method with ammonium molibdate. All statistical analyses were performed by means of the SPSS version 10.0 software for Windows. Stepwise linear discriminant analysis simplified the system to the following variables: Na, K, Mg, number of leucocytes, triglycerides, GPT, glucose, albumin, retinol and folic acid; and 90.1% (86.4% after crossvalidation) of correct classification was obtained. Representing the first and second discriminant functions, the control groups were well separated from opiate addicts. Applying linear discriminant analysis on several biochemical and haematological parameters, the opiate addicts could clearly be differentiated from the control individuals, and a tendency to differentiate the opiate addicts in MMTP and in detoxification treatment was observed.

Simultaneous determination of tocotrienols, tocopherols, retinol, and major carotenoids in human plasma.

Epidemiologic evidence suggests that the concentrations of antioxidant vitamins in human plasma may play an important role in numerous chronic diseases, such as cancer and cardiovascular disease. However, methods for simultaneous measurement of these antioxidants are scarce. We developed and validated a new HPLC method for simultaneous determination of these vitamers in human plasma that uses a novel column-switching approach. The new method uses liquid-liquid extraction and isocratic separation with two monomeric C(18) columns maintained at 35 and 4 degrees C coupled with ultraviolet-visible and fluorometric detection. This method could separate 14 vitamers and 3 internal standards within 27 min. No additional modifier was required; the mobile phase was acetonitrile-methanol (65:35 by volume), and the flow rate was 1 mL/min. For photodiode array detection, the detection limits (signal-to-noise ratio >3) were 0.02 mg/L for beta-carotene, lutein, zeaxanthin, and canthaxanthin; 0.01 mg/L for all-trans-retinol, beta-cryptoxanthin, alpha-carotene, and lycopene; and 0.1 mg/L for all tocopherols and tocotrienols. The detection limit was at least 25-fold lower (0.004 mg/L) when fluorometry was used for measurement of delta-, gamma-, and alpha-tocotrienol and delta-tocopherol compared with ultraviolet detection. The recovery and imprecision of the assay were generally >90% and <10%, respectively. This new method separates a wide range of fat-soluble antioxidant vitamins in human plasma, including six carotenoids, three isoforms of tocotrienols and tocopherols (delta-, gamma-, and alpha-), and all-trans-retinol. The overall findings suggest that our method is faster, more sensitive, and more comprehensive than existing methods.

Development of a simplified method for the simultaneous determination of retinol, alpha-tocopherol, and beta-carotene in serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A new and simple method for the determination of fat-soluble vitamins (retinol, alpha-tocopherol, and beta-carotene) in human serum was developed and validated by using liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS-MS). Different solvent mixtures were tested to obtain deproteinization and extraction of the analytes from the matrix. As a result, a volume of 240 microL of a 1:1 (v/v) ethanol/ethyl acetate mixture added to 60 microL of serum was found to be suitable for both protein precipitation and antioxidants solubilization, giving the best recovery for all three analytes. Deproteinized samples (20 microL) were injected after dilution, without the need for concentration or evaporation to dryness and reconstruction of the sample. Vitamins were separated on a C-8 column using a 95:5 (v/v) methanol/dichloromethane mixture and ionized in the positive-ion mode; detection was performed in the selected-reaction monitoring mode. Linearity of the LC-APCI-MS-MS method was established over 5 orders of magnitude for retinol and alpha-tocopherol, whereas in the case of beta-carotene it was limited to 4 orders. Lower limits of quantitation were 1.7, 2.3, and 4.1 nM for retinol, alpha-tocopherol, and beta-carotene, respectively. Serum concentrations of retinol, alpha-tocopherol, and alpha+beta-carotene determined in a group of healthy volunteers were 2.48, 38.07, and 0.50 microM, respectively, in samples collected in winter ( n=122) and 2.69, 45.88, and 0.90 microM during summer ( n=66).

Simultaneous Determination of Retinoic Acid, Retinol, and Retinyl Palmitate in Ram Plasma by Liquid Chromatography

A liquid chromatographic method is described for the simultaneous determination of retinoic acid, retinol, and retinyl palmitate in ram plasma. Samples of 0.2 mL are extracted with 2‐propanol‐dichloromethane, the extracts are centrifuged, the supernatants are collected, evaporated, reconstituted in mobile phase, and analyzed on a C8 column using two consecutive isocratic elutions with methanol tetrahydrofuran‐acetate buffer. Detection is performed at 350 and 325 nm using wavelength change during the run. The method exhibits analytical characteristics well within acceptable limits. Overall recoveries were 73.7% for retinoic acid, 90.2% for retinol, and 87.7% for retinyl palmitate. Precision values, expressed as % relative standard deviation, were in the range of 1.16–6.18%, while limits of detection were in the range of 0.3–10.0 ng/mL for all analytes.

Method for the Simultaneous Determination of Retinol and β-Carotene Concentrations in Human Tissues and Plasma

To understand differential tissue distribution of retinoids and carotenoids, as it might influence biological processes in humans, we developed and demonstrated a method for measuring them in selected human tissues. The method includes internal standards and a secondary reference standard to eliminate the need for external standard calibration and to minimize sample-handling errors. Tissues were digested (saponified) in ethanolic KOH. Retinol and β-carotene were extracted with organic solvent containing internal standards. Analytes were separated using isocratic liquid chromatography and quantified at 325 nm for retinol and 450 nm for β-carotene. Plasma was analyzed in a similar way but without saponification. Retinal-O-ethyloxime and β-apo-12′-carotenal-O-t-butyloxime served as internal standards. Plasma, breast, and fat from breast surgery patients and colon, liver, muscle, and fat from colon surgery patients were analyzed. Within-day relative standard deviations (RSDs) for plasma were <0.04 for β-carotene and <0.03 for retinol, between-day RSDs were <0.05 for β-carotene and <0.04 for retinol. Saponification ensured complete extraction of retinol and β-carotene and removal of triglycerides that “foul” chromatographic columns. It seems retinol and β-carotene concentrations in tissues and blood of cancer patients are the same or higher than those in corresponding tissues of patients without these cancers.

Fast determination of retinol and α-tocopherol in plasma by LC

A fast, selective and economical method for the determination of retinol and α-tocopherol is presented. Both vitamins are separated by high-performance liquid chromatography (HPLC) in less than 4 min using an isocratic elution with methanol. The robustness of the method was checked in real samples, obtaining relative standard deviation lower than 3%. The described method was satisfactorily applied to serum samples proceeding of patients enrolled in a methadone maintenance treatment program. The retinol concentrations in the serum of these patients fell into the normal interval of concentrations; however, the serum α-tocopherol contents were higher than the normal values.

Liver retinol and carotenoid concentration of rats experimentally infected with trypanosoma brucei

The acute phase response to infection has been reported to include a decline in the vitamin A status of the infected host in some disease, and the application of vitamin A supplementation in the management of these disease conditions had led to significant reductions in the severity, morbidity and mortality associated with them. Also cellular and tissue injury in trypanosome infections has partly been attributed to oxidative stress and depletion of some systemic antioxidants. This study investigated the liver retinol and carotenoid concentration of rats experimentally infected with Trypanosoma brucei. Results of the liver retinol determination showed that T. brucei infection led to a progressively significant (P < 0.01) depletion of liver retinol concentration (body vitamin A status) of infected rats from day 5 to day 25 post-infection. The decline was found to be significantly directly proportional to the degree of hepatomegaly (r = 0.778, p < 0.05) and splenomegaly (r = 0.778, p < 0.05), but not significantly related to the level of parasitaemia (r = 0.407, p > 0.05). Results of the liver carotenoids determination showed a gradual depletion of liver carotenoids from day 5 post-infection, being most severe from day 10 to day 20 post-infection when the mean liver carotenoids of infected rats was significantly (p < 0.0 1) lower than that of uninfected ones. These findings suggest prospects for trials on vitamin A supplementation in the management of T. brucei infections, and support the assertion that oxidative stress plays a significant role in cellular injury in trypanosome infections. (Tropical Veterinarian: 2002 20(1): 1-7)

Effects of β-carotene and vitamin A on oval cell proliferation and connexin 43 expression during hepatic differentiation in the rat

The effects of β-carotene and vitamin A administrations were evaluated in an in vivo model of hepatic cell differentiation. For this purpose, male Wistar rats received β-carotene (70 mg/kg of body weight), vitamin A (10 mg/kg of body weight) or corn oil (control group), by gavage and at every other day during the entire experimental period. After 4 consecutive weeks of treatment, the animals were submitted to the AAF/PH model of hepatic cell differentiation (6 × 20 mg of AAF [2-acetylaminofluorene]/kg of body weight and partial hepatectomy) and killed on different days following the surgery (until day 16 after hepatectomy). Liver samples were collected for determination of β-carotene, retinol and retinyl palmitate concentrations, for histopathological (hematoxilin-eosin) examination, for immunohistochemical detection of glutathione S-transferase, as well as for the evaluation of connexin 43 (a structural protein of gap junctions of oval cells) expression by northern blot analysis. Compared to controls, the oval cell proliferation peaks (observed by histopathological examination and immunohistochemistry) and connexin 43 expression peaks, were postponed to later days after hepatectomy, in a similar way in β-carotene and vitamin A treated animals. Compared to the other experimental groups, the vitamin A treated group showed an increase in connexin 43 expression. It was concluded that β-carotene and vitamin A modulated oval cell proliferation and connexin 43 expression, delaying both events. These findings suggest that β-carotene and vitamin A can modulate the hepatic differentiation process in vivo.

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.

Simultaneous determination of all-trans, 9-cis, 13-cis retinoic acid and retinol in rat prostate using liquid chromatography-mass spectrometry.

Since retinoic acid (RA) and RA receptors are key developmental regulators during organogenesis, they might participate in the abnormal development of the prostate caused by early estrogen exposure. In order to test this assumption, a sensitive analytical method that can differentiate 9-cis, 13-cis, and all-trans RA in small tissue samples ( approximately 8 mg) is required. Since retinol is the metabolic precursor to RA, simultaneous quantification of retinol would also provide valuable information. Here, we report a liquid chromatography-mass spectrometry method for simultaneous determination of retinol and 9-cis, 13-cis, and all-trans RA in rat prostate. Mass spectrometric signal responses for RA were compared using positive ion atmospheric-pressure chemical ionization (APCI) and electrospray, as well as positive ion and negative ion APCI. Positive ion APCI was selected for all subsequent analysis for its better sensitivity, and to provide simultaneous determination of retinol and RA. Ventral prostate tissue samples were homogenized and extracted following simple protein precipitation without derivatization. Baseline separation of 9-cis, 13-cis, and all-trans RA standards was obtained by using a non-porous silica C18 column. Selected ion monitoring of the ions m/z 301 and m/z 269 was carried out for mass spectrometric quantitative analysis. The ion of m/z 301 corresponded to the protonated molecule of RA, whereas the ion of m/z 269 corresponded to loss of water or acetic acid from the protonated molecule of retinol or the internal standard retinyl acetate respectively. The method has a linear response over a concentration range of at least three orders of magnitude. The limit of quantitation was determined to be 702 fmol all-trans RA injected on-column. The method showed excellent intra- and inter-assay reproducibility and good recovery, and is suitable for analyzing RA and retinol in small tissue samples (approximately 8 mg).

Rapid high-performance liquid chromatographic method for the simultaneous determination of retinol, α-tocopherol and β-carotene in human plasma and low-density lipoproteins

A reversed-phase HPLC method with diode-array detection was used to simultaneously determine retinol, α-tocopherol and β-carotene in human plasma and low-density lipoproteins. An aliquot of sample was de-proteinized with ethanol containing α-tocopherol acetate as internal standard, and the analytes were extracted twice with hexane. The solvent was evaporated to dryness under a stream of nitrogen and the residue was redissolved in methanol to be injected directly into the HPLC system. A multiple solvent system based on methanol, butanol and water at a flow-rate of 2 ml/min and held at 45°C provided clear separation of these compounds in only 8 min. The method showed good linearity, precision and accuracy for all compounds. Owing to its simplicity, this method may be useful in routine clinical and epidemiological work.

ANTIOXIDANT STATUS OF U.S. BIATHLETES DURING ALTITUDE TRAINING

The goal of this study was to assess antioxidant status during a 10-day cross-country skiing training camp. Participants included four U.S. Biathlon Team members (3 male, 1 female, age = 23 yr, VO2max = 70.4 ml/kg/min male, 54 ml/kg/min female). Training took place at 2,100 m and consisted of 2–4 hours per day of cross-country skiing at approximately 60–70% VO2max. Blood samples for the determination of retinol, beta-carotene, alphatocopherol, gamma-tocopherol as well as the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were measured before and after exercise on days 1 and 10. Plasma vitamin measures were determined by HPLC analysis and the GSH/GSSG was determined colorimetrically by OXIS Research, Portland OR. A comparison between the athletes' vitamin status and published normative values indicated that all athletes were within normal ranges on both days 1 and 10 for all antioxidant vitamins measured. A decrease in the GSH/GSSG ratio is a sensitive marker of oxidative stress and indicates a decrease in the reductive capacity of the red blood cell. On day 10, the post-exercise GSH/GSSG ratio (153:1) was significantly lower than the pre-exercise value (241:1, p = .016). Also, the GSH/GSSG ratio following exercise on day 10 (153:1) was lower than the post-exercise value on day 1 (202:1, p = .06). These data indicate a change in redox status after an exercise bout and over 10 days of aerobic training at altitude. Although antioxidant vitamin status was normal throughout the training camp, the redox status was shifted toward an oxidative state. Oxidative stress is often regarded as a negative consequence of training, however, it is possible that it may provide a stimulus for physiological adaptation.

Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, α-tocopherol, 25-hydroxyvitamin D 3 and 25-hydroxyvitamin D 2 in human plasma with photodiode-array ultraviolet detection

A new rapid and sensitive high-performance liquid chromatographic method using 0.5 ml of plasma has been developed for the simultaneous determination of retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D 2 and 25-hydroxyvitamin D 3. The eluate was monitored with a photodiode-array detector with two fixed wavelengths (267 nm for vitamin D, 292 nm for α-tocopherol and retinol). For all compounds, including internal standards, the method provides extraction recoveries greater than 81%. Detection limits were equal to or lower than 1.5 μg/l for the 4 vitamins. Linearity of standards was excellent ( r>0.999 in all cases). Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was 3 4 7.7 for all compounds and the inter-day-assay C.V. was <9.2% except for the lower concentrations of 25-hydroxyvitamin D 3, 25-hydroxyvitamin D 2 and α-tocopherol (10.8, 11.8 and 11.9, respectively). The important properties of the present method are its ease of use, its rapidity, since sample preparation was achieved in 15 min and all the compounds were eluted in less than 15 min, and its small sample volume required (=0.5 ml), which enables it to be used in pediatric practice.

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.

Simultaneous determination of retinal, retinol and retinoic acid (all- trans and 13- cis) in cosmetics and pharmaceuticals at electrodeposited metal electrodes

The electrochemical behaviors of retinoids on glassy carbon and electrodeposited metal electrodes were investigated in various aqueous or nonaqueous containing supporting electrolytes, respectively. Retinal, retinol, all- trans-retinoic acid (all- trans-RA ) and 13- cis-retinoic acid (13- cis-RA) can be separated at the thin-film lead deposited glassy carbon electrode in 0.1 M tetrabutylammonium hydroxide. The electroreduction process is applied for the simultaneous quantitative determination of retinoids in cosmetics and pharmaceuticals. Comparison with results obtained from high performance liquid chromatography shows good agreement.

Improved method of determining retinol and retinyl palmitate in rat liver and serum by high-performance liquid chromatography.

In this study, we examined the effect of certain analytical procedures to determine the best method of recovering the ingested retinoids, specifically retinol and retinyl palmitate, from rat liver and serum. In this experiment, the best extraction solvent for retinol was n-hexane and that for retinyl palmitate was ethyl acetate. The best results were obtained using a mobile phase (n-hexane-diethyl ether, 76:24, v/v) as the sample solvent in the assay for liver retinol, similarly, chloroform as the sample solvent in the assay for serum retinol, and for liver retinyl palmitate, the best sample solvent was methanol-toluene (5: 5, v/v). The assayed values of retinol and retinyl palmitate measured in ethanol (0.125% BHT added) and extraction solvent (0.025% BHT added) were significantly higher than those when no BHT was added to the ethanol and extraction solvent. The determination methods for extracting retinol and retinyl palmitate from the liver varied according to the conditions layed out above. Simultaneous determination of retinol and retinyl palmitate has been illustrated in previous papers by various authors: however, we found that the individual determination of retinol and retinyl palmitate was necessary to accurately assay each retinoid.

Significance of simultaneous determination of serum and seminal plasma alpha-tocopherol and retinol in infertile men by high-performance liquid chromatography.

High-performance liquid chromatography was used for the simultaneous determination of alpha-tocopherol and retinol in serum and semen of 40 subfertile men. The serum levels of the two vitamins were significantly higher in serum than in semen (3- to 10-fold) (P < 0.001). The mean alpha-tocopherol levels were higher in the serum and semen of men with normal sperm parameters (20 +/- 5 and 5 +/- 4 mumol L-1) than those with oligozoospermia (10 +/- 4 and 3 +/- 2 mumol L-1), azoospermia (8 +/- 3 and 3 +/- 1 mumol L-1) and asthenozoospermia (9 +/- 6 and 3 +/- 2 mumol L-1) (P < 0.002). Mean retinol levels in serum were similar in men with normal sperm parameters (2.4 +/- 2 mumol L-1) as in those with defective sperm parameters such as oligozoospermia (2.5 +/- 2 mumol L-) and asthenozoospermia (2.1 +/- 1.0 mumol L-) (P = 0.15), but significantly lower in men with azoospermia (1.3 +/- 0.3 mumol L-1) (P < 0.05). The alpha-tocopherol:retinol ratio was significantly higher in semen than in serum of men with normal sperm parameters (11.5) and those with asthenozoospermia (10.3) compared with ratios in those with oligozoospermia (8.3) and azoospermia (6.3). This may be related to reduced antioxidant activity in sperm dysfunction as a result of lipid peroxidation, from increased generation of reactive oxygen species.

Simultaneous determination of serum retinol and α- and γ-tocopherol levels in type II diabetic patients using high-performance liquid chromatography with fluorescence detection

This paper presents a simple reversed-phase high-performance liquid chromatographic method for the simultaneous determination of retinol, and α- and γ-tocopherols in human serum using a fluorescence detector. For chromatographic separation a binary gradient was used: phase A; acetonitrile–butanol (95:5); phase B; water, at a flow-rate of 1.5 ml/min. Serum retinol, and α- and γ-tocopherol levels were measured in patients with non-insulin-dependent diabetes mellitus. Small sample requirement, good reproducibility and sensitivity make this method useful for the determination of the serum levels of these compounds in patients with diabetes mellitus.