Development and validation of an ion-pair liquid chromatographic method for the quantitation of sodium cromoglycate in urine following inhalation

An ion-pair liquid high-performance chromatography method with solid-phase extraction for measuring urinary concentrations of sodium cromoglycate following inhalation has been developed and validated. Sodium cromoglycate was extracted from urine on a 100-mg phenyl cartridge (Isolute, Jones Chromatography) and then quantified on a 25-cm C₈ Spherisorb 5 μm stationary phase with a mobile phase of methanol-0.045 M phosphate buffer-0.05 M dodecyl triethyl ammonium phosphate (550:447.6:2.4, v/v) pH 2.3, at 0.85 ml min⁻¹ using nedocromil sodium as an internal standard and UV detection at 238 nm. The inter- and intra-day reproducibilities were 8.33 and 13.63%, respectively, at 0.25 mg l⁻¹. The limit of determination for sodium cromoglycate was 0.25 mg l⁻¹ (with a signal-to-noise ration of greater than 10:1). Following oral and inhaled administration of 20 mg of sodium cromoglycate to eight healthy volunteers, the mean and S.D. of sodium cromoglycate excreted in the urine at 0.5, 1 and 24 h post-dose were 0.02, 0.05 and 0.33%, and 0.16, 0.30 and 1.55% of the dose, respectively. The urinary recovery of sodium cromoglycate at 0.5 and 1 h following inhalation can therefore be used to compare the amount of drug reaching the respiratory tract using different sodium cromoglycate inhaled products or inhalation methods.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 μg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9–2.1 and 1.1–6.4%, respectively, at concentrations ranging between 5 and 200 μg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

Internal standard high-performance liquid chromatography method for the determination of obidoxime in urine of organophosphate-poisoned patients

Obidoxime is an antidote approved for reactivation of inhibited acetylcholinesterase in organophosphate poisoning. HPLC methods were described for its determination in blood or aqueous solutions but not for the determination in urine. Since data for renal obidoxime excretion ranged from 2.2 to 84% of administered dose in healthy volunteers depending on the route of administration and little is known about pharmacokinetics of obidoxime in severely intoxicated patients we developed an internal standard (HI 6) reversed-phase HPLC method for determining obidoxime in urine. The mobile phase consisted of methanol, the counter ion 1-heptane sulfonic acid and tetrabutylammonium phosphate, the stationary phase involved a 5 μm reversed-phase column (125×4 mm). Obidoxime was detected spectrophotometrically at 288 nm. The limit of quantification (LOQ) was 1 μM, the limit of detection (LOD) 0.5 μM. Linear calibration curves were obtained in a concentration range from 1 to 1000 μM. Intra- and inter-day precision C.V.s were below 4%. Accuracy was 95.9% in the LOQ range. Using this method, we were able to quantify obidoxime in urine of an organophosphate poisoned patient. Based on this data we calculated that 58% of the administered dose was excreted in the urine.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Enantioselective determination of selfotel in human urine by high-performance liquid chromatography on a chiral stationary phase after derivatization with 9-fluorenylmethyl chloroformate

An analytical method for the enantioselective determination of selfotel in human urine has been developed and validated. The method is based on high-performance liquid chromatography and utilizes CGS 20005 (a selfotel analog) as the internal standard. Urine samples were derivatized in situ with o-phthalic dicarboxaldehyde–3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate (FMOC). Chromatographic separations of the FMOC derivatives of selfotel enantiomers and the internal standard were achieved using a column switching system consisting of an Inertsil ODS-2 column (75×4.6 mm I.D., 5 μm) and a Chiralcel OD-R column (250×4.6 mm I.D., 10 μm). The composition of the mobile phase was acetonitrile–0.1 M phosphate buffer, pH 2.50 (35:65) for the Inertsil ODS-2 column and acetonitrile–0.1 M phosphate buffer, pH 2.00 (35:65) for the Chiralcel OD-R column. The analytes were monitored using fluorescence detection at an excitation wavelength of 262 nm and an emission wavelength of 314 nm. The limit of quantification (LOQ) for this method is 0.25 μg/ml for each selfotel enantiomer. The method was successfully utilized to determine preliminary selfotel stereospecific pharmacokinetics.     

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58–86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.     

Oral platelet aggregation inhibitor Ro 48-3657: Determination of the active metabolite and its prodrug in plasma and urine by high-performance liquid chromatography using automated column switching

A sensitive and highly automated high-performance liquid chromatography (HPLC) column-switching method has been developed for the simultaneous determination of the active metabolite III and its prodrug II, both derivatives of the oral platelet inhibitor Ro 48-3657 (I), in plasma and urine of man and dog. Plasma samples were deproteinated with perchloric acid (0.5 M), while urine samples could be processed directly after dilution with phosphate buffer. The prepared samples were injected onto a pre-column of a HPLC column switching system. Polar plasma or urine components were removed by flushing the precolumn with phosphate buffer (0.1 M, pH 3.5). Retained compounds (including II and III) were backflushed onto the analytical column, separated by gradient elution and detected by means of UV detection at 240 nm. The limit of quantification for both compounds was 1 ng/ml (500 μl of plasma) and 25 ng/ml (50 μl of urine) for plasma and urine, respectively. The practicability of the new method was demonstrated by the analysis of about 6000 plasma and 1300 urine samples from various toxicokinetic studies in dogs and phase 1 studies in man.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250×4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 μM CDA (S/N=3). The linear range found applying the optimized conditions was 0.2 to 200 μM. The intra- and inter-day assay precision (R.S.D.) of CDA (10 μM) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C₁₈ solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5 · 10⁻⁴ M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

High-performance liquid chromatographic–electrochemical assay for the quantitation of BMS-181885 in monkey plasma

A high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantitation of BMS-181885 (I), an anti-migraine agent, in monkey plasma. The assay involved a solid-phase extraction of I and BMY-46317 (internal standard; I.S.) on a 1-ml cyano cartridge using the automatic solid-phase extraction cartridge (ASPEC) system. Immediately following the conditioning of the cyano column (3 ml of methanol and 2 ml of 1% glacial acetic acid), plasma (0.25 ml) was loaded on to the column. The column was then washed with a 3 ml of 0.1 M ammonium acetate buffer (pH 6). The final elution of the analytes was performed using 2 ml of methanol. The eluate was then evaporated to dryness (gentle stream of nitrogen at 40°C) and the residue was dissolved in the mobile phase and injected on to a YMC basic column (15 cm×4.6 mm; 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 0.1 M ammonium acetate at pH 6–acetonitrile–methanol (70:20:10, v/v) was used as the mobile phase. Standard curves, with a lower limit of quantitation of 2 ng/ml of I were linear (r²≥0.998; range: 2–50 ng/ml). Based on the analysis of the quality control (QC) samples, the assay was both accurate and precise. The stability of I was established following freeze–thaw cycles and storage at or below −20°C. The extraction recovery of I from monkey plasma was about 82%. The validated assay method was applied to determine the pharmacokinetics of I in monkeys following a single 1 mg/kg intravenous dose.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Determination of trovafloxacin,a new quinolone antibiotic,in biological samples by reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C₁₈ column with a mobile phase consisting of 0.04 M H₃PO₄-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 μg/ml and the calibration curves were linear over a concentration range of 0.1 to 20..0 μg/ml (r² = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 μg/ml. Human serum samples could be stored for up to 12 months at −20°C and urine samples could be stored up to 18 months at −80°C.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d₃ and 7-AF-d₃ were used as internal standards. The drugs were separated on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d₃ and F-d₃), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d₃), 330 (3-OHF). The limits of detection were: 0.2 μg/l for F and 7-AF, 1 μg/l for N-DF and 3-OHF. The method was linear in the range 1–500 μg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC–APCI-MS seems to be a method of choice for these compounds.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (±)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 μM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid β-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (?)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 × 10^?4 and 0.177 × 10^?4 mol/mol protein. Multipie dosing increased irreversible binding 3- to 4-fold. © 1993 Wiley-Liss, Inc.     

Sensitive semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681, the active parent drug of a new oral carbapenem antibiotic,DZ-2640, in human plasma and urine using a column-switching system as sample clean-up procedure

DZ-2640 is a new oral carbapenem antibiotic having a dihydro-pyrroloimidazole ring as a side chain and a pivaloyloxymethyl (POM) ester prodrug of DU-6681, the active parent compound. A simple and sensitive column-switching semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681 in human plasma and urine has been developed. Human plasma was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was filtered through an Ultrafree C3GV. The resulting filtrate was injected without further cleanup onto the HPLC system. Human urine was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was directly injected onto the HPLC system. The analyte was detected by monitoring the column effluent with UV light at a wavelength of 300 nm, which resulted in the limit of quantitation of 0.008 μg/ml of plasma and 0.32 μg/ml of urine. Calibration curves were linear in the range of 0.008 to 5.85 μg/ml in plasma and 0.32 to 104.4 μg/ml in urine. The present methods showed greatly increased sensitivity for DU-6681 compared to conventional HPLC methods and also showed satisfactory recovery, selectivity, precision, and accuracy. Stability studies showed that 1 M MOPS buffer (pH 7.0) acted as a stabilizer. In plasma and urine diluted with equal volume of the buffer, DU-6681 showed good stability at −80°C for up to 4 weeks with no significant loss of the drug.