High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir,saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid–liquid extraction with diethyl ether from 500 μl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate–acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C₁₈ column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20 000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30 000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.     

Stereoselective determination of R(−)- and S(+)-MK-571, a leukotriene D4 antagonist,in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(−)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D₄ antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine—acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine—pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r²>0.999) for concentrations of enantiomers of 1 from 0.05 μg/ml, the lowest quantitation limit, up to 2.5 μg/ml. Intra-day coefficients of variation at 0.05 μg/ml were 2.4% for the R(−)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(−)- and S(+)-1 were 2.6 and 3.6%, respectively. The utilit of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Quantitative high-performance liquid chromatography-based detection method for calphostin C,a naturally occurring perylenequinone with potent antileukemic activity

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca²⁺-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250×4 mm LiChrospher 100, RP-18 (5 μm) in conjunction with a 4×4 mm LiChrospher 100, RP-18 guard column (5 μm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile–water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05–40 μM for calphostin C in 50 μl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 μl plasma was 0.02 μM at a signal-to-noise ratio of ∼3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Simultaneous analysis of disopyramide and quinidine in plasma by high-performance liquid chromatography

A new reversed-phase high-performance liquid chromatographic method allowing simultaneous measurement of plasma concentrations of disopyramide and quinidine is described. Disopyramide and quinidine were separated on a reversed-phase column using 0.05 M phosphate buffer (pH 3.0)—acetonitrile (73:27, v/v), as mobile phase and the peaks were monitored by UV absorbance at the wavelengths of 254 and 325 nm. The drugs were extracted from alkaline plasma with chloroform containing the internal standard. The organic phase was evaporated to dryness and the residue was redissolved in a small volume of the mobile phase before analysis by high-performance liquid chromatography. The method is convenient and reliable in routine monitoring of both drugs.     

High-performance liquid chromatographic determination of clindamycin in human plasma or serum: application to the bioequivalency study of clindamycin phosphate injections

This paper presents an assay of clindamycin phosphate injection in human plasma or serum. A 0.5-ml volume of plasma was used with the internal standard, propranolol. The sample was loaded onto a silica extraction column. The column was washed with deionized water and then eluted with methanol. The eluates were evaporated under nitrogen gas. The residue was reconstituted with the mobile phase and injected onto the high-performance liquid chromatographic system: a 5-μm, 25 cm×4.6 mm I.D. ODS2 column was used with acetonitrile, tetrahydrofuran and 0.05 M phosphate buffer as the mobile phase and with ultraviolet detection at 204 nm. A limit of quantitation of 0.05 μg/ml was found, with a coefficient of variation of 11.6% (n=6). The linear range is between 0.05 and 20.00 μg/ml and gives a coefficient of determination (r²) of 0.9992. The method has been successfully applied to the bioavailability study of two commercial preparations of clindamycin phosphate injection (300 mg each) in twelve healthy adult male volunteers.     

Quantitation of itraconazole in rat heparinized plasma by liquid chromatography–mass spectrometry

A liquid chromatographic–mass spectrometric (LC–MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized plasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 ml) with acetonitrile containing nefazodone as an internal standard (I.S.), a 50-μl aliquot of the supernatant was mixed with 100 μl of 10 mM ammonium formate (pH 4.0). An aliquot of 25 μl of the mixture was injected onto a BDS Hypersil C₁₈ column (50×2 mm; 3 μm) at a flow-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was detected in single ion monitoring (SIM) mode. Standard curves were linear (r²≥0.994) over the concentration range of 4–1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of ITZ was 96%. The validated assay was applied to a pharmacokinetic study of ITZ in rats following administration of a single dose of itraconazole (15 mg/kg).     

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.     

Determination of quinapril and quinaprilat by high-performance liquid chromatography with radiochemical detection,coupled to liquid scintillation counting spectrometry

Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase high-performance liquid chromatographic procedure with radiochemical detection, coupled to liquid scintillation counting spectrometry. Quinapril and quinaprilat were measured in perfusate and urine after pretreatment with acetonitrile and subsequent centrifugation. Perfusate ultrafiltrate was used as collected. Two quinapril diketopiperazine metabolites, PD 109488 and PD 113413, were separated chromatographically from quinapril, quinaprilat, and from each other. Assay performance for quinapril and quinaprilat was assessed by examining precision and accuracy of the assay over four days. Using a 100-μl sample volume, the limit of quantitation for both ³H-quinapril and ³H-quinaprilat (sp. act. ≈ 2.0 μCi/μg) was 1 ng/ml.     

Simple,rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 μm LiChrosorb-RP18 column (250×4 mm I.D.) at 50°C. The mobile phase was 6 mM phosphoric acid–acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 μg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 μg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 μl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.     

Determination of the quinidine analog, 7′-trifluoromethyldihydrocinchonidine-2HCl in plasma and urine by high-performance liquid chromatography

A rapid and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiarrhythmic quinidine analog, 7′-trifluoromethyldihydrocinchonidine-2HCl ([I]-2HCl) in plasma and urine. The overall recovery of [I] from plasma was 86 ± 9% with a sensitivity limit of detection of 0.2 μg/ml.The assay involves extraction of [I] into benzene-methylene chloride (9:1) from plasma or urine made alkaline with 0.1 N sodium hydroxide (pH 13) and saturated sodium chloride, the residue of which is dissolved in methylene chloride, an aliquot of which is analyzed by HPLC using adsorption chromatography on silica gel with UV detection at 254 nm. The mobile phase composed of methylene chloride-methanol-conc. ammonium hydroxide (95.5:4:0.5) yields baseline resolution of quinidine used as the internal (reference) standard, compound [I] and dihydroquinidine, a common contaminant in quinidine.The assay was applied to the analysis of plasma and urine samples taken from a dog administered a single 20 mg/kg dose via intravenous and oral routes. The stability of [I] in human plasma for up to 37 days of storage at ?17°C was also demonstrated.     

Purification of human brain metallothionein by organic and reversed-phase high-performance liquid chromatography under acidic conditions

A simplified high-performance liquid chromatographic method for the detection of metallothioneins, notably metallothionein-III, has been developed. In order to purify metallothionein, differential acetone precipitation at 50% (v/v) and at 80% (v/v) was employed on a 20% normal human brain homogenate. The reconstituted pellet was injected into a C₁₈ microbore reversed-phase HPLC column, equilibrated with 0.1% trifluoroacetic acid, and developed at a flow-rate of 800 μl/min with a linear gradient from 0% to 60% acetonitrile in 0.094% trifluoroacetic acid for 60 min. Western blots indicated that metallothioneins-I and II eluted at 16% acetonitrile and metallothionein-III eluted at 37% acetonitrile.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

Elimination of caffeine interference in high-performance liquid chromatographic determination of cotinine in human plasma

A high-performance liquid chromatographic method with ultraviolet photometric detection for the determination of cotinine in human plasma was described. The use of a 30-cm reversed-phase column and of a mobile phase consisting of water—methanol—0.1 M sodium acetate—acetonitrile (72:21:5.6:1.4, v/v), pH 4.1, eliminated caffeine interference. A simplified solid-phase extraction procedure was also performed for plasma samples.     

Analysis of cisapride in neonatal plasma using high-performance liquid chromatography with a base-stable column and fluorescence detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the prokinetic drug cisapride is described. Alkalinised samples of plasma (100 μl) were extracted with 1.0 ml of 10% (v/v) isopropanol in chloroform, dried, redissolved in mobile phase and injected. Chromatography was performed at 20°C by pumping a mobile phase of acetonitrile (370 ml) in pH 5.2, 0.02 M phosphate buffer (630 ml) at 1.0 ml/min through a C₈ Symmetry column. Cisapride and the internal standard were detected by fluorescence monitoring at 295 nm (excitation) and 350 nm (emission), and were eluted 5 min and 8 min, respectively, after injection. Calibration plots in bovine serum albumin (3% w/v) were linear (r > 0.999) from 5 to 250 ng/ml. Intra-day and inter-day precision (C.V.) was 9.5%, or less, and the accuracy was within 5.5% of the nominal concentration over the range 8–200 ng/ml. Total assay recovery was above 82%. Endogenous plasma components, major cisapride metabolite (norcisapride), and other durgs used in neonatal pharmacotherapeutics did not interfere.     

High-performance liquid chromatographic determination of the β2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization

A sensitive high-performance liquid chromatographic method has been developed for the determination of the β₂-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid–liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher^® 100 RP 18 and a LiChrospher^® RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.     

High-performance liquid chromatographic assay of (±)-ethopropazine and its enantiomers in rat plasma

Two high-performance liquid chromatographic (HPLC) methods are described for determination of (±)-ethopropazine (ET) in rat plasma. After deproteination and liquid–liquid extraction, assay of (±)-ET was performed using either a C₁₈ column (non-stereospecific assay) or an (α-R-naphthyl)ethylurea column (stereospecific assay). The UV detection was at 250 nm. Mean recovery was >85%. Both assays demonstrated excellent linear relationships between peak height ratios and plasma concentrations; quantitation limits were ≤25 ng/ml, based on 100 μl rat plasma. Accuracy and precision were <17% with both methods. Both methods were applied successfully to the measurement of ET plasma concentrations in rats given the drug intravenously.     

Quantitative determination of naproxen in plasma by a simple high-performance liquid chromatographic method

A high-performance liquid chromatographic method for the determination of naproxen in plasma is described. The technique is based on the single extraction of the drug from acidified plasma with chloroform using 2-naphthalene acetic acid as internal standard. The chromatographic system consisted of a column packed with Spherisorb ODS (5 μm); the mobile phase was acetonitrile—phosphoric acid (pH 3) (45:55, v/v).The method can accurately measure plasma naproxen concentrations down to 1 μg/ml using 100 μl of sample, with no interference from endogenous compounds. The coefficients of variation of the method at 120 μg/ml and 1 μg/ml are 2.8 and 21.6%, respectively, and the calibration curve is linear. The method described is very suitable for routine clinical and pharmacokinetic studies.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.