Quantitation of individual tocopherols in plasma, platelets, lipids, and livers by high-performance liquid chromatography

A rapid, sensitive procedure is described for the analysis of the tocopherols (α-, β-, γ-, and δ-) in plasma, platelets, lipids, and liver using high-performance liquid chromatography and fluorometric detection. Excellent recoveries of these tocopherols in plasma were obtained - greater than 90%. Separation and quantitation of the four tocopherols required as little as 0.2 ml plasma.     

Lipid and fatty acid composition of striped bass (Morone saxatilis) larvae during development

Changes in lipid class, fatty acid composition, protein, and dry and wet weights of fertilized eggs and developing larvae of striped bass (Morone saxatilis) fed with the live food, Artemia, were investigated. A decrease of wet and dry weights and moisture was observed at the beginning of the larval stage. Larvae regained the original moisture level, and wet and dry weights increased steadily after feeding. Total lipids decreased from 190 μg/egg in fertilized eggs to 151 μg/egg during hatching and increased after feeding. When total lipid contents were expressed as a percentage of larval dry weight, a decline of lipid did not occur until after feeding. Total protein, on the other hand, increased right after feeding, but there was some variation between days. Polar lipids increased significantly from 20 μg/egg at the egg stage to 199 μg/larva at 26 days post-hatching (DPH), 2 days before the onset of metamorphosis, while neutral lipids declined from 175 μg/egg to 80 μg/larva during the same time period. Wax/steryl esters decreased from 150 μg/egg in fertilized eggs to 32 μg/larva at 26 DPH. Triacylglycerols dropped from 21 μg/egg to 15 μg/larva before feeding and increased gradually after feeding. In contrast, the level of cholesterol increased 2–3-fold. There was a significant increase of phospholipids, particularly phosphatidylcholine in larvae after feeding. The fatty acid composition of fish larvae was significantly influenced by the diet, Artemia. There was an indication of catabolism of endogenous eicosapentaenoic and docosahexaenoic acids during metamorphosis.     

In vitro cleavage of paracetamol glucuronide by human liver and kidney β-glucuronidase: determination of paracetamol by capillary electrophoresis

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human β-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 μM (38 ng/ml) and a limit of quantitation of 1 μM (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human β-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human β-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.     

Determination of the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C₁₈, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N₄-acetyl-5-methylhydroxysulfamethoxazole (N₄SOH) and sulfamethoxazole-N₁-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by β-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N₄-acetylsulfamethoxazole (N₄), SOH, N₄-hydroxysulfamethoxazole (N₄OH), N₄SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N₄ 0.10 μg/ml; N₄SOH 0.13 μg/ml; N₄OH 0.18 μg/ml; SOH 0.20 μg/ml; and Sgluc 0.39 μg/ml. In urine the quantitation limits are: N₄ and N₄OH 1.4 μg/ml; S 1.5 μg/ml; N₄SOH 1.9 μg/ml; SOH 3.5 μg/ml; and Sgluc 4.1 μg/ml. The method was applied to studies with healthy subjects and HIV positive patients.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

Inhibitory effects of Schizandrae Fructus on eotaxin secretion in A549 human epithelial cells and eosinophil migration

Eosinophilia have been implicated in a broad range of diseases, most notably allergic conditions (e.g. asthma, rhinitis and atopic dermatitis) and inflammatory diseases. These diseases are characterized by an accumulation of eosinophils in the affected tissue. Defining the mechanisms that control the recruitment of eosinophil is fundamental to understanding how these diseases progress and identifying a novel target for drug therapy. Accordingly, this study was conducted to evaluate the regulatory effect of Schizandrae Fructus (SF) on the expression of eotaxin, an eosinophil-specific chemokine released in respiratory epithelium following allergic stimulation, as well as its effects on eosinophil migration.To accomplish this, human epithelial lung cells (A549 cell) were stimulated with a combination of TNF-α (100 ng/ml) and IL-4 (100 ng/ml) for 24 h. The cells were then restimulated with TNF-α (100 ng/ml) and IL-1β (10 ng/ml) to induce the expression of chemokines and adhesion molecules involved in eosinophil chemotaxis for another 24 h. Next, the samples were treated with various concentrations of Schizandrae Fructus (SF) (1, 10, 100, 1000 μg/ml) or one of the major constituents of SF, schizandrin (0.1, 1, 10, 100 μg/ml), after which following inhibition effect assay was performed triplicates in three independence.The levels of eotaxin in secreted proteins were suppressed significantly by SF (100 and 1000 μg/ml, p<0.01) and schizandrin (10 and 100 μg/ml, p<0.01). In addition, SF (1, 10, 100 and 1000 μg/ml) decreased mRNA expression levels in A549 cells significantly (p<0.01). Eosinophil recruitment to lung epithelial cells was also reduced by SF, which indicates that eotaxin plays a role in eosinophil recruitment. Furthermore, treatment with SF suppressed the expression of another chemokine, IL-8 (0.1 and 1 μg/ml SF, p<0.01), as well as intercellular adhesion molecule-1 (10 and 100 μg/ml SF, p<0.01) and vascular cell adhesion molecule-1 (0.1 and 1 μg/ml SF, p<0.05), which are all related to eosinophil migration. Taken together, these findings indicate that SF may be a desirable medicinal plant for the treatment of allergic diseases.     

Measurement of 5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol) in human plasma and urine by high-performance liquid chromatography

Three high-performance liquid chromatographic methods are described for the detection of the novel antifolate anticancer drug (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol): one with fluorometric detection and two with detection by UV absorbance. An assay for plasma lometrexol using UV detection (288 nm) and reversed-phase chromatography was developed, with a quantitation limit of 0.2 μg/ml and linearity up to 10 μg/ml. This assay was modified for measurement of lometrexol in urine, with a quantitation limit of 2 μg/ml and linearity up to 25 μg/ml. An alternative assay for plasma lometrexol using derivatization and fluorescence detection (excitation at 325 nm, emission at 450 nm) was also developed, which proved twenty-fold more sensitive (quantitation limit of 10 ng/ml) than the UV assay, and which was linear up to 250 ng/ml. The fluoremetric method requires sample oxidation with manganese dioxide prior to analysis, and uses ion-pair chromatography with tetramethylammonium hydrogensulphate as an ion-pair reagent. All assays use a similar preliminary solid-phase extraction method (recovery as assessed by UV absorption >73%), with C10-desmethylene lometrexol added for internal standardisation. Each assay is highly reproducible (inter-assay precision in each assay is <10%). Applicability of the fluorescence-based assay to lometrexol in plasma and the UV-based assay lometrexol in urine is demonstrated by pharmacokinetic studies in patients treated as part of a Phase I clinical evaluation of the drug.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Serum α- and γ-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, β-carotene, α-carotene, cryptoxanthin, lutein, lycopene, α-tocopherol, and γ-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk’s Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P<0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 μg/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P<0.05) higher for Seven Seas dolphins (16.4 μg/ml) than for Hawk’s Cay (13.0 μg/ml) and free-ranging dolphins (12.5 μg/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and β-carotene) while the captive animals did not. Additional carotenoids (lycopene, α-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Determination of gabapentin in plasma by high-performance liquid chromatography

A rapid and simple method for determination of the novel antiepileptic compound gabapentin [1-(aminomethyl)cyclohexaneacetic acid] in plasma is described. Blank human plasma was spiked with gabapentin (1.0–10.0 μg/ml) and internal standard [1-(aminomethyl)-cycloheptaneacetic acid; 5.0 μg/ml]. Individual samples were treated with 2 M perchloric acid, centrifuged and then derivatised with o-phthalaldehyde-3-mercaptopropionic acid. Separation was achieved on a Beckman Ultrasphere 5 μm reversed-phase column with mobile phase consisting of 0.33 M acetate buffer (pH 3.7; containing 100 mg/l EDTA)-methanol-acetonitrile (40:30:30, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 330 and 440 nm, respectively. The calibration curve for gabapentin in plasma was linear (r=0.9997) over the concentration range 1.0–10.0 μg/ml. Recovery was seen to be 90%. The inter- and intra-assay variations for three different gabapentin concentrations were 10% throughout. The lower limit of quantitation was found to be 0.5 μg/ml. Chromatography was unaffacted by a range of commonly employed antiepileptic drugs or selected amino acids.     

The differential effect of Eriobotrya japonica hydrophilic leaf extract on cytokines production and modulation

Stimulating or modulating the release of cytokines by immunomodulators or immunostimulating agents is an attractive mode for treating several diseases such as viral infections. For instance, patients with viral infections may be in need of increasing or inducing T helper 1 (Th1) or proinflammatory cytokines, which ultimately activate T cytotoxic and Natural killer lymphocytes to kill virally infected cells. Of these agents, we found that Eriobotrya japonica hydrophilic leaf extract (EJHE) can induce and modulate cytokines in dose-dependent manner. Twenty-four hour exposure of increasing concentrations of EJHE increased significantly (p < 0.001) the production of IFN-γ and TNF-α, from PHA+LPS-stimulated whole blood. However, the production of IFN-γ and TNF-α plateaued at high EJHE concentrations (10–100 μg/ml). No significant changes in the production of IL-10 were seen. In addition, EJHE at 1 and 10 μg/ml reversed significantly (p < 0.01) the inhibitory effect of hydrocortisone on the IL-12 p70, IFN-γ and TNF-α production from PHAS+LPS stimulated whole blood. Without PHA and LPS, EJHE was found to induce significantly (p < 0.001) IFN-γ, IL-12 p70, TNF-α, and IL-10 from whole blood culture in concentration dependent manner. The maximum induction of IFN-γ, IL-12 p70, and TNF-α by EJHE was at 1 and 10 μg/ml. On the other hand, IL-10 induction kept increasing even at the highest concentration used (100 μg/ml) of EJHE. Furthermore, intra-peritoneal injection of EJHE into mice increased significantly serum cytokines level mainly at 10 and 100 μg/ml. Two-hour post i.p. injection, EJHE increased serum IFN-γ, TNF-α, and IL-10 to 750, 1000, and 250 pg/ml, respectively. However, 24 h post i.p. injection, the levels of TNF-α, and IL-10 were similar to basal levels but IFN-γ levels were 200 pg/ml. These results indicate that EJHE induces proinflammatory and Th1 cytokines in concentration dependent manner and the effect of this induction should be studied further in viral models to check the efficacy of such cytokine induction.     

Rapid high-performance liquid chromatographic determination of serum gabapentin

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 μg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 μg/ml, 14.8% were below 2 μg/ml and 12.7% above 10 μg/ml. The mean±S.E. of GBP in 283 patients was 5.38±0.23 μg/ml. Peak concentrations of more than 15 μg/ml and trough levels as low as 0.1 μg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 μg/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α from the Day 15 guinea-pig uterus superfused . These findings indicate that the high output of PGF_2α from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 gmg/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 gmg/ml but not 1 μg/ml) significantly reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20–50% by oestradiol (10 μg/ml). The addition of oestradiol (10 μg/ml) and progesterone together (10 gmg/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 μg/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 gmg/ml) reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

Background

Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6^th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells.

Results

Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl₂ and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl₂ gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6.

Conclusions

As a conclusion, secondary metabolite contents were increased by cadmium chloride application and sampling time, while dry cell weights was reduced by cadmium chloride treatments.     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.