Orotic acid protects pancreatic ß cell by p53 inactivation in diabetic mouse model.

Impairment of pancreatic ß cells is a principal driver of the development of diabetes. Restoring normal insulin release from the ß cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 ß cells and subsequently reduced ß cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of ß cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained ß cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes.

The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Exploring pathogenesis in subjects with subjective Tinnitus having kidney deficiency pattern in terms of Traditional Chinese Medicine based on serum metabolic profiles.

OBJECTIVE: To investigate the metabolic pathogenesis in subjects with subjective tinnitus (ST) having kidney deficiency pattern (KDP) (ST/KDP) in terms of Traditional Chinese Medicine. METHODS: Three groups of subjects, including healthy individuals, subjects with ST/KDP, and subjects who were healthy initially and then developed ST/KDP one year later (healthy ¡ú ST/KDP), were recruited for this study. Serum metabolic profiles of all subjects were analyzed using ultra-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry. The metabolic characteristics of the ST/KDP subjects were determined, and the corresponding biomarkers were predicted. The metabolomics data from the healthy ¡ú ST/KDP subjects were collected for further verification. RESULTS: Twelve metabolites in the ST/KDP subjects were different from those of the healthy control subjects. Of these metabolites, according to the prediction, except for octanoic acid, other metabolites might characterize ST/KDP. Ten metabolites at the outcome ST/KDP stage were different from those at the initial (control) stage. Through the comparison of these metabolites with the predicted metabolites, five common metabolites, including upregulated glutamate, serotonin, orotic acid and 8-oxoguanine, as well as downregulated taurine, were found. These common metabolites were significantly associated with canonical pathways including calcium signaling, ¦Ã-aminobutyric acid (GABA) receptor signaling, purine and pyrimidine biosynthesis, taurine biosynthesis, and serotonin receptor signaling. CONCLUSION: The metabolic pathogenesis in ST/KDP subjects was characterized by upregulated glutamate, serotonin, orotic acid and 8-oxoguanine, as well as downregulated taurine, additionally, perturbations of calcium signaling, GABA receptor signaling, purine and pyrimidine biosynthesis, taurine biosynthesis, and serotonin receptor signaling.

A comparative pharmacokinetic and tolerability analysis of the novel orotic acid salt form of tenofovir disoproxil and the fumaric acid salt form in healthy subjects.

A novel orotic acid salt form of tenofovir disoproxil (DA-2802) was developed and is expected to replace the fumaric acid salt form. The pharmacokinetic (PK) characteristics and tolerability profiles of DA-2802 were compared to those of tenofovir disoproxil fumarate (TDF, Viread®) in healthy subjects. A randomized, open-label, single-dose study was conducted in 36 healthy subjects using a two-treatment, two-period, and two-sequence crossover design. Subjects received a single oral dose of 319 mg DA-2802 or 300 mg TDF, during each period, with a 7-day washout. Serial blood samples were collected pre-dosing and up to 72 hours post-dosing in each period, for determination of serum tenofovir concentration, which was measured by ultra-performance liquid chromatography-tandem mass spectrometry. A non-compartmental method was used to obtain PK parameters of tenofovir. For comparison between the two tenofovir disoproxil salts, the 90% confidence intervals (90% CIs) of geometric mean ratios of DA-2802 to TDF for the maximum concentration (Cmax) and the area under the concentration-time curve to the last quantifiable concentration (AUC0-t) were determined. The tolerability profiles of tenofovir were assessed by evaluation of adverse events and vital signs, physical examination, ECG, and clinical laboratory tests. The serum tenofovir concentration-time profiles of DA-2802 or TDF were comparable in 32 subjects who completed the study. In both profiles, a two-compartmental elimination with first-order elimination kinetics in the terminal phase was reported in a few subjects, showing a secondary peak in the initial phase of elimination. The geometric mean ratio (90% CI) of DA-2802 to TDF was 0.898 (0.815-0.990) for Cmax and 0.904 (0.836-0.978) for AUC0-t. There were no clinically significant findings in the tolerability assessments. DA-2802 showed comparable PK characteristics and tolerability profiles to TDF.

Elevated plasma dihydroorotate in Miller syndrome: Biochemical, diagnostic and clinical implications, and treatment with uridine.

BACKGROUND: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis. METHODS: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems. RESULTS: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO. CONCLUSION: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening.

Development of an assay to simultaneously measure orotic acid, amino acids, and acylcarnitines in dried blood spots.

BACKGROUND: Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS. METHODS: Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA-MS/MS) with negative-mode ionization, requiring <2 min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid. RESULTS: We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75-85% at multiple levels of enrichment. Precision was also comparable to traditional FIA-MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2 µmol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects. CONCLUSION: The assays described here quantify orotic acid in DBS using a simple extraction and FIA-MS/MS analysis procedures that can be implemented into current NBS protocols.

Towards newborn screening for ornithine transcarbamylase deficiency: fast non-chromatographic orotic acid quantification from dried blood spots by tandem mass spectrometry.

BACKGROUND: Orotic acid (OA) is the key parameter in the detection of ornithine transcarbamylase deficiency (OTC-D). Inclusion of OA into newborn screening compatibility with existing analytical procedures is necessary. METHODS: OA was eluted from dried blood spots with methanol containing deuterated [1,3-(15)N2] OA as internal standard. Quantification by tandem mass spectrometry was accomplished without chromatographic separation. Samples were measured in MRM mode for the masses m/z 155.1 → 111 for OA and 157.1 → 113 for d2 OA. RESULTS: OA was determined in a wide range of concentrations with high precision, LOD and LOQ being 0.21 and 0.65 µmol/L, respectively. Values correlated well with those obtained after chromatography. Pretreatment of samples with HCl-butanol regularly used for acylcarnitine measurement did not significantly affect quantitative results. Inclusion of the new method into the standard newborn screening procedure did not alter the results for acylcarnitines or amino acids; the total time per analysis, however, was increased from 1.15 to 1.85 min. OA levels of 707 unaffected newborns ranged from 0.28 to 3.73 µmol/L. Five newborns with OTC-D showed concentrations of 89.7-211.1 µmol/L. In newborns with severe citrullinaemia we found values in the range of 4.99-127.7 µmol/L. CONCLUSIONS: This new method can be used as a standalone measurement of OA but it can also easily be implemented into standard newborn screening techniques as a useful supplement. In this case the method allows detection of newborns with OTC deficiency without an extra analytical run.

Spectrophotometric simultaneous determination of orotic acid, creatinine and uric acid by orthogonal signal correction-partial least squares in spiked real samples.

An orthogonal signal correction-partial least squares (OSC-PLS) method was developed for the simultaneous spectrophotometric determination of orotic acid (OA), creatinine (CRE), and uric acid (UA) in spiked real samples. By multivariate calibration methods, such as PLS regression, it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. The effect of OSC used to remove the information unrelated to the target variables is studied. In this study, the calibration model is based on absorption spectra in the 220-320 nm rang for 36 different mixtures of OA, CRE and UA. Calibration matrices contained 1.74-47.00 of OA, 1.13-33.95 of CRE, and 1.68-28.58 of UA in µg/ml. The number of principal component for OA, CRE, and UA with OSC were 3, 4, and 4, and 4, 6, and 5, without OSC, respectively. The evaluation of the prediction errors for the prediction set reveals that the OSC-treated data give substantially lower root mean square error of prediction (RMSEP) values than the original data. The RMSEP for OA, CRE, and UA with OSC were 0.69, 0.20, and 0.53 and 0.80, 0.69, and 0.73 without OSC, respectively. The proposed method was applied for the simultaneous determination of OA, CRE, and UA in spiked biological fluids with satisfactory results.

Reference intervals for orotic acid in urine, plasma and dried blood spot using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of some hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study was aimed to establish age related reference intervals of OA in urine, and for the first time in plasma, and dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years. The quantification of OA was performed by a previously implemented method, using a stable isotope dilution with 1,3-[(15)N(2)]-orotic acid and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The method has proved to be sensitive and accurate for a quantitative analysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of creatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups aged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers (from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more stable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (µM) also resulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and 4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (µM) were significantly lower in subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59, 0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS of 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these disorders.

Orotic acid quantification in dried blood spots and biological fluids by hydrophilic interaction liquid chromatography tandem mass spectrometry.

Orotic acid (ORA) is an intermediate metabolite in the pathway of pyrimidine nucleotides; its urinary excretion is useful to diagnose the hereditary orotic aciduria and some hyperammonemic inherited defects of urea cycle enzymes and amino acid transporters. ORA analysis is based on stable isotope dilution by GC-MS or LC-MS/MS methods. We developed a fast assay that measures the ORA in dried blood spots (DBS), plasma and urine using hydrophilic interaction LC-MS/MS. Within- and between-day analytical imprecision (CV%) of three quality control levels, in plasma, DBS and urine, ranged from 0.8 to 14.1%, while the inaccuracy ranged from -13.5 to 9.4%. In healthy children (n=20), ORA concentrations were less than 0.69 microM in plasma, less than 0.82 microM in DBS and from 0.2 to 1.4 mmol/mol of creatinine in urine. A patient with citrullinemia showed ORA levels of 133 microM in plasma and 39 microM in DBS. A patient with hyperammonemia-hyperornithinemia-homocitrullinemia (HHH) syndrome presented a urinary ORA level of 9.1 mmol/mol of creatinine. The method is potentially able to discriminate affected patients from reference subjects; the clinical validation should be expanded on a higher number of patients.

Ghrelin and orotic acid increased in subclinical mastitis.

Hormone ghrelin and orotic acid accelerate wound healing as well as controlling inflammation and immunity. We have, therefore, investigated the serum and milk levels of ghrelin and orotic acid in dairy cows with (n = 21) or without (n = 21) subclinical mastitis. Acylated and des-acylated ghrelin as well as orotic acid concentration were detected by using high performance liquid chromatography (HPLC). The results revealed that ghrelin level in milk and serum was significantly higher in dairy cows with subclinical mastitis than that of dairy cows without subclinical mastitis. This was also the case when the orotic acid concentrations in dairy cows with subclinical mastitis were compared with those dairy cows without subclinical mastitis. In conclusion, ghrelin and orotic acid occur in particularly high concentrations in subclinical mastitis, and might, therefore, be required in greater amounts for tissue repair and may be also used as a indicator for subclinical mastitis.

Japanese black cattle with orotic aciduria detected by gas-chromatography/mass-spectrometry.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

Two step derivatization for the analyses of organic, amino acids and glycines on filter paper plasma by GC-MS/SIM.

A rapid dried-filter paper plasma-spot analytical method was developed to quantify organic acids, amino acids, and glycines simultaneously in a two-step derivatization procedure with good sensitivity and specificity. The new method involves a two-step trimethylsilyl (TMS) - trifluoroacyl (TFA) derivatization procedure using GC-MS/ selective ion monitoring (GC-MS/SIM). The dried-filter paper plasma was fortified with an internal standard (tropate) as well as a standard mixture of distilled water and methanol. Methyl orange was added to the residue as an indicator. N-methyl-N-(trimethylsilyl-trifluoroacetamide) and N-methyl-bis-trifluoroacetamide were then added and heated to 60 degrees C for 10 and 15 min to produce the TMS and TFA derivatives, respectively. Using this method, the silylation of carboxylic functional groups was carried out, which was followed by the trifluoroacyl derivatization of the amino functional group. The derivatives were analyzed by GC-MS/SIM. A calibration cure showed a linear relationship for the target compounds between concentrations of 10-500 ng/mL. The limit of detection and quantification on a plasma spot were 10-90 ng/mL (S/N=9) and 80-500 ng/ mL, respectively. The correlation coefficient ranged from 0.938 and 0.999. When applied to the samples from positive patients, the method clearly differentiated normal subjects from the patients with various metabolic disorders such as PKU, MSUD, OTC and a Propionic Aciduria. The new developed method might be useful for making a rapid, sensitive and simultaneous diagnosis of inherited organic and amino acid disorders. In addition, this method is expected to be an alternative method for screening newborns for metabolic disorders in laboratories where expensive MS/MS is unavailable.

Ornithine aminotransferase deficiency: diagnostic difficulties in neonatal presentation.

We describe two unrelated cases of ornithine aminotransferase (OAT) deficiency with rare neonatal presentation of hyperammonaemia. The diagnosis in the neonatal presentation of OAT deficiency is hampered as hyperornithinaemia is absent. Enzyme and mutation studies confirmed the diagnosis. OAT deficiency should be included in differential diagnosis of neonatal hyperammonaemia.

Dietary arginine supplementation enhances the growth of milk-fed young pigs.

This study was conducted to determine the effect of dietary arginine supplementation on the growth of artificially reared piglets. The pigs (n = 24; 7 d old) were removed from sows to a nursery facility and assigned randomly to 1 of the 3 treatments representing diets supplemented with 0, 0.2, or 0.4% L-arginine (on the basis of milk replacer powder). Each milk feeder was assigned to 1 dietary treatment. Fresh liquid milk replacer (18.6% dry matter) was provided daily ( approximately 0800 h) to piglets. Body weights of piglets were measured and jugular venous blood samples were obtained for metabolite analysis at d 7, 14, and 21 of age. Food intake did not differ between control and arginine-supplemented piglets [66.7 vs. 69.5 g dry matter/(kg body wt. d)]. Compared with control piglets, dietary supplementation with 0.2 and 0.4% L-arginine dose dependently increased (P < 0.05) plasma concentrations of arginine by 30 and 61%, and decreased (P < 0.05) plasma concentrations of ammonia by 20 and 35%, and those of urea by 19 and 33%, respectively. Dietary supplementation with 0.4% L-arginine also increased (P < 0.05) plasma concentrations of insulin and growth hormone by 24-27% in piglets, compared with controls. Between 7 and 21 d of age, the supplementation of 0.2 and 0.4% L-arginine to piglets enhanced (P < 0.05) average daily weight gain by 28 and 66%, and body weight by 15 and 32%, respectively, compared with control piglets. Collectively, both the metabolic and growth data demonstrate unequivocally that arginine is deficient in milk-fed young pigs and that this arginine deficiency represents a major obstacle to maximal growth in piglets.

Plasma concentrations and renal clearance of orotic acid in argininosuccinic acid synthetase deficiency.

Argininosuccinic acid synthetase deficiency (ASD) is a rare disorder of urea cycle metabolism, with pronounced citrullinemia and orotic aciduria being characteristic biochemical features. To further investigate the role of plasma orotic acid and its possible use for monitoring the metabolic status in ASD, we determined plasma orotic acid, amino acid, and ammonium levels in plasma samples collected over a period of 3 years from a patient who is now 8 years of age. Orotic acid plasma concentrations varied widely from less than 1 micromol/l to more than 60 micromol/l. The renal clearance of orotic acid was eightfold the glomerular filtration rate, thus supporting an active mechanism underlying the excretion of this pyrimidine. Data obtained during a metabolic crisis yielded a statistically significant linear correlation of orotic acid plasma levels with those of glutamine and ammonium, which are generally accepted for assessment of the successful treatment of this disorder. Our data revealed no advantage of plasma orotic acid concentrations over the established amino acids (glutamine and arginine) and ammonium for determining acute treatment responses. Since several effects of high levels of orotic acid have been described in mammals, further research is necessary to assess a possible contribution of orotic acid to the pathogenesis of ASD and the use of plasma orotic acid levels in the long-term monitoring of these patients.

Clinical and biochemical heterogeneity in females of a large pedigree with ornithine transcarbamylase deficiency due to the R141Q mutation.

A large family with ornithine transcarbamylase deficiency due to mutation R141Q was ascertained through a propositus who presented with acute neonatal hyperammonemic coma. Of 13 females at risk, 11 were evaluated clinically and had laboratory studies performed. Seven were found to be heterozygous for the mutation. Of these seven, five had chronic clinical symptoms and two were asymptomatic. None of the heterozygotes had elevated plasma ammonia on random testing. Of the five symptomatic females, three had markedly elevated plasma glutamine levels on random testing, while two had levels in the upper range of normal. Plasma citrulline and arginine levels were somewhat lower in the symptomatic individuals but still within the normal range. Five heterozygotes who were tested had either spontaneous orotic aciduria or elevated orotic acid following ingestion of allopurinol, whereas one unaffected female and one unaffected male had normal allopurinol tests. A higher than expected proportion of female heterozygous for the R141Q mutation were clinically and biochemically symptomatic but remained undiagnosed for many years. Plasma glutamine determination and allopurinol testing should be performed in females who present with a combination of relatively non-specific symptoms detailed in this report.

The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency.

The sparse fur (spf/Y) mouse was evaluated as a model for studying gene therapy in ornithine carbamoyltransferase deficiency (OCTD), the most common inborn error of urea synthesis. Previous studies have defined a number of biochemical characteristics of this animal model that are analogous to the human disease: OCTD in liver, elevated ammonium and glutamine, low citrulline and arginine in plasma, elevated urinary orotic acid excretion, neurochemical alterations and responsiveness to alternative pathway therapy. In this study, metabolic flux, survival, behavior and learning of these animals were examined in preparation for a trial of gene therapy. We found that, as has been previously reported, OCT activity in liver ranged from 10 to 20% of control. Yet, stable isotope studies using 15N ammonium chloride to follow ureagenesis in vivo showed 55% of normal urea synthetic capacity. This suggests that partial correction with gene therapy may be sufficient to normalize urea synthesis. Although it has been suggested that liver OCTD and its consequent metabolic effects normalize without treatment by adulthood in the spf/Y mouse, we did not find this to be the case. We documented that the spf/Y mouse had a markedly decreased lifespan (< 10% of normal) and remained runted throughout life. In terms of behavior, the spf/Y mice had evidence of decreased learning in a passive avoidance task that was not attributable to alterations in activity. These clearly definable metabolic and behavioral abnormalities suggest that the spf/Y mouse should prove a useful model for studying the efficacy of gene therapy in OCTD.

Determination of orotic acid in serum by high-performance liquid chromatography.

A simple method for the determination of orotic acid in serum is described. The analyses were carried out using a strong anion-exchange column. Orotic acid was separated isocratically with 0.8 M formic acid (pH 2.8, adjusted with 10 M NaOH)-methanol (65:35, v/v) at a flow-rate of 1.0 ml/min. Absorbance at 275 nm was recorded for quantification. Validation yielded a detection limit of 0.07 micrograms/ml and a quantification limit of 0.25 micrograms/ml. The maximum within-day and day-to-day coefficients of variation were less than 6%. The method has the advantage that samples can be easily prepared.