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%.     

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 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.     

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.     

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.     

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.     

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.     

Determination of quinolone antibiotics in growth media by reversed-phase high-performance liquid chromatography

A simple, accurate, precise, and versatile high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of three quinolone antibiotics in Mueller–Hinton broth. The fluoroquinolone agents studied were ciprofloxacin, ofloxacin, and sparfloxacin; other quinolone agents have been identified using this method but not validated in this matrix (levofloxacin, clinafloxacin, temafloxacin, and trovafloxacin). In addition, several other biological growth mediums have been investigated (human serum, human urine, Todd–Hewitt growth media, Ensure enteral feeding solution, and Haemophilus growth media). This method uses UV detection (280 nm), a simple, one-step protein precipitation extraction, and separation using a C₁₈ column with an isocratic, ion-pairing mobile phase. An appropriate internal standard was obtained by using another quinolone antibiotic of differing retention time. The calibration curves were linear (r²≥0.999) over a concentration range of 0.0625–20.0 μg/ml with a lower limit of quantification of 0.1 μg/ml. The intra-day and inter-day coefficients of variation were less than 15%.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Determination of Quinidine and its major metabolites by high-performance liquid chromatography

A specific and precise assay, capable of quantitating in human plasma simultaneously but separately quinidine, dihydroquinidine and the quinidine metabolites 2′-quinidinone, 3-OH-quinidine and a third metabolite found — tentatively identified as the product formed by rearrangement of quinidine-N-oxide — is reported. The assay uses a normal phase high-performance liquid chromatographic (HPLC) system with a variable-wavelength UV detector at 235 nm and has a limit of sensitivity at approximately 20 ng/ml. The mobile phase consists of hexanes—ethanol—ethanolamine (91.5:8.47:0.03). A 2-ml plasma sample is worked up by adding primaquine base as an internal standard and extracting with ether—dichloromethane—isopropanol (6:4:1). The organic extract is evaporated and the residue reconstituted in 100—600 μl of mobile phase and an aliquot injected onto the column.Comparison of this procedure with the Edgar and Sokolow (dichloroethane) extraction—fluorescence procedure and with the Cramer and Isaksson (benzene) double extraction—fluorescence assay indicates that both fluorescence procedures give quinidine concentrations up to 2.3 times those determined by HPLC. These discrepancies were shown to be due to carry-over of metabolites and some extraneous background fluorescence.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C₁₈ reversed-phase column using isocratic elution at 40°C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r²>0.99) was observed within the calibration ranges studied: 37.4–299.3 μg/ml for PRM, 26.4–211.2 μg/ml for PB, 12.5–100.2 μg/ml for p-HO-PB and 12.1–97.0 μg/ml for PEMA. Repeatability was in the range 3.1–6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.     

Determination of amikacin in microlitre quantities of biological fluids by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

Pre-column derivatization of amikacin with 1-fluoro-2,4-dinitrobenzene in 25 μl of guinea pig plasma or human serum produced a stable chromophore which was measured by UV detection after rapid separation on normal-phase or reversed-phase high-performance liquid chromatography systems. The reversed-phase system, selected for routine analysis due to instability of the normal-phase column, consisted of an Ultrasphere-ODS C18 column preceded by a guard column, and used acetonitrile—water (68:32) as the mobile phase. A high degree of linearity was found in the range of 2—64 μg/ml with a coefficient of variation averaging less than 5%.     

Determination of metformin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the hypoglycemic agent metformin is described. Acidified samples of plasma were deproteinated with acetonitrile, washed with dichloromethane and the resulting supernatant injected. Chromatography was performed at 40°C by pumping a mobile phase of acetonitrile (250 ml) in pH 7, 0.03 M diammonium hydrogen phosphate buffer (750 ml) at a flow-rate of 1 ml/min through a silica column. Metformin and the internal standard (atenolol) were detected at 240 nm and were eluted 7.8 and 6.8 min, respectively, after injection. No endogenous substances were found to interfere. Calibration curves were linear (r>0.999) from 10 to 2000 ng/ml. The absolute recovery of both metformin and atenolol was greater than 76%. The detection limit and limit of quantitation were 2.5 and 10 ng/ml, respectively. The intra- and inter-day precision (C.V.) was 12%, or less, and the accuracy was within 6.2% of the nominal concentration. This method is suitable for clinical investigation and monitoring metformin concentration.