Column liquid chromatographic determination of clozapine and N-desmethylclozapine in human serum using solid-phase extraction

A 0.5-ml aliquot of a serum sample, after the addition of a 50-μl aliquot of a 5 μg/ml solution of amoxapine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifugated. The supernatant is applied to a 1-ml BondElut C₁₈ silica extraction column-conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 15-μl aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₈ silica particles and eluted at ambient temperature with a mobile phase of 0.1% tetramethylammonium perchlorate-acetonitrile (73:27, v/v) adjusted to pH 4.2 with 10% perchloric acid. The peaks are detected with an absorbance detector at 245 nm.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

Optimization of a column liquid chromatographic procedure for the determination of plasma salbutamol concentration

A reversed-phase column liquid chromatographic procedure with fluorescence detection for the determination of salbutamol in plasma is described. A 1-ml aliquot of the sample, after the addition of bamethan as the internal standard, is passed through a Bond Elut silica extraction column. The column is selectively washed to remove neutral, acidic, and weakly basic compounds. The desired compounds are eluted with a 1-ml aliquot of methanol. The eluate is evaporated under vacuum at ambient temperature and the residue is reconstituted in 40 μl of the mobile phase which contains octanesulfonic acid as the ion-pairing reagent. The entire extract is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm octylsilica particles. Peaks are detected with a fluorescence detector (excitation WAVELENGTH = 275 nm, emission WAVELENGTH = 310 nm). In the resulting chromatogram, salbutamol and the internal standard give sharp peaks that are well resolved from the extraneous peaks. The procedure allows the quantitation of salbutamol down to 0.2 ng/ml.     

Optimization of the determination of 4-aminopyridine in human serum and urine by column liquid chromatography

Two convenient reversed-phase column liquid chromatographic procedures are described for the determination of 4-aminopyridine in human serum and urine. A 0.5-ml aliquot of serum after the addition of a 0.5-ml solution of 4-(aminomethyl)pyridine in 0.1M Na₂HPO₄ as the internal standard is passed through a 1-ml BondElut C₁₈ silica extraction column. The column is selectively washed to remove acidic, neutral and weakly basic compounds. The desired compounds are eluted with a 0.3-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 10-μl aliquot of the eluate is infected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₁₈ silica particles that is eluted at ambient temperature with a mobile phase containing octanesulfonic acid as the ion-pairing agent. The peaks are monitored at 263 nm. A 0.25-ml aliquot of urine or 0.5 ml of serum is mixed with N-propionylprocainamide as the internal standard and subjected to benzoylation by Schotten Baumann reaction. The reaction mixture is adjusted to pH 5.5–6 and extracted with a BondElut C₁₈ extraction column. An aliquot of the eluate is chromatographed at ambient temperature with a mobile phase containing tetramethylammonium perchlorate. The peaks are monitored at 278 nm.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

High-performance liquid chromatographic determination of methoxsalen in plasma after liquid—solid extraction

An improved method suitable for the determination of 8-methoxypsoralen in the range 50–1500 ng/ml in the plasma of psoriatic patients undergoing PUVA (psoralens and long-wave ultraviolet light) therapy is proposed. A 5-ml aliquot of plasma containing sodium citrate as anticoagulant was centrifuged, griseofulvin was added as internal standard and the sample was denatured with acetonitrile. The supernatant was applied to C₁₈ cartridges and 8-methoxypsoralen was eluted with methanol. The evaporated eluate was reconstituted in the mobile phase for high-performance liquid chromatography (HPLC) and applied to the HPLC column: mobile phase, acetonitrile—0.01 M phosphoric acid (34:66); flow-rate, 1 ml/min; temperature, 40°C; column, Spherisorb 5 ODS, 100 mm × 4.6 mm I.D., 5 μm particle size; UV detection at 248 nm; detection limit, 15 ng/ml of plasma.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the determination of azosemide and its metabolite, M1, in human plasma and urine and rabbit blood and tissue homogenates. The methods involved deproteinization of the biological samples: 2.5 volumes of acetonitrile were used for the determination of azosemide and 1 volume of saturated Ba(OH)₂ and ZnSO₄ for that of M1. A 50-μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column in each instance. The mobile phases employed were 0.03 M phosphoric acid—acetonitrile (50:40, v/v) for azosemide and 0.03 M phosphoric acid/0.2 M acetic acid—acetonitrile (83:17, v/v) for M1. The flow-rate was 1.5 ml/min in both instances. The column effluent was monitored by ultraviolet detection at 240 and 236 nm for azosemide and M1, respectively. The retention times for azosemide and M1 were 6.0 and 8.3 min, respectively. The detection limits for both azosemide and M1 in both human plasma and urine were 50 ng/ml. The coefficients of variation of the assay were generally low (below 11.0%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or other diuretics tested were observed.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C₈ preparative column with 6% acetonitrile—45% methanol—5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-μm Spin-X cartridges, applied to a reversed-phase C₁₈ column, and eluted in a 0–50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzylamiloride.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.     

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid–liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 μl of mobile phase and 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10–9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.     

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.     

Sensitive high-performance liquid chromatographic assay for motexafin gadolinium and motexafin lutetium in human plasma

We present new HPLC methods for the quantitation in human plasma of two investigative metallotexaphyrin agents, motexafin gadolinium (Gd-Tex) and motexafin lutetium (Lu-Tex). Each assay uses: the other texaphyrin analogue as an internal standard; protein precipitation with acetonitrile:methanol (50:50, v/v); an ODS reversed-phase column; an isocratic mobile phase of 100 mM ammonium acetate, pH 4.3:acetonitrile:methanol (59:21:20, v/v/v); and absorbance detection at 470 nm. The Gd-Tex assay has a lower limit of quantitation (LLOQ) of 0.01 μM and is linear between 0.01and 30 μM. The Lu-Tex assay has an LLOQ of 0.1 μM and is linear between 0.1 and 30 μM. The assays are suited for in vivo preclinical studies and clinical trials because they require minimal amounts of plasma, are sensitive, and involve a 30-min run time. These assays are important tools for evaluating the potential of Gd-Tex and Lu-Tex as a radiation enhancer and photosensitizer, respectively.     

High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

Method for separation and determination of lactone and hydroxy acid forms of a new HMG CoA reductase inhibitor (RG 12561) in plasma

The new drug RG 12561 (I) is a lactone that is undergoing clinical evaluation for its cholesterol lowering effect based on potent HMG CoA reductase inhibitory activity displayed by its open hydroxy acid form. To determine the dispositional characteristics of the drug, a method was developed for determination of the two forms in plasma. A 0.25-ml aliquot of plasma was deproteinized with 0.5 ml of methanol, and the lactone was extracted with hexane—ethyl acetate (75:25, v/v). The methanolic plasma was then acidified followed by extraction of the hydroxy acid with hexane—ethyl acetate. The extracts were dried, reconstituted and analyzed by isocratic, reversed-phase high-performance liquid chromatography using ultraviolet absorbance at 254 nm. The separations were performed utilizing a C₁₈ column with mobile phase consisting of acetonitrile, 2-propanol and 0.1 M acetate buffer (pH 5), the proportions of which differed depending on the form of drug analyzed. The method was found to be selective and a quantitation limit of 50 ng/ml was established. Validation studies demonstrated that the method was sufficiently accurate and precise for determining disposition of the drug in the dog.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

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.     

Determination of amphotericin B in human serum by liquid chromatography

A rapid reversed-phase high-performance liquid chromatographic method with a 30-mm long column is described for assaying amphotericin B in serum. After deproteinization of serum samples with methanol, the supernatant was injected onto a reversed-phase C₁₈ column, using 2.5 mM Na₂EDTA-acetonitrile (70:30, v/v) as the mobile phase. Amphotericin B was eluted at 1.5 min. Calibration plot of the peak area against concentration was linear from 0.05 to 25 μg/ml (C.V. of 3%). Within-day and day-to-day imprecision (C.V.) ranged between 1.33% and 3.61%. The application was evaluated in 55 serum samples from patients treated with amphotericin B.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.