A field-adapted HPLC method for determination of amodiaquine and its metabolite in whole blood dried on filter paper

A reversed-phase high performance liquid chromatographic method was developed and validated for the quantitative determination of amodiaquine (AQ) and its metabolite desethylamodiaquine (DAQ) in whole blood collected on filter paper. The structure analogue 4-(4-dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. Upon collection, blood was added to 10% phosphoric acid in a 1:1 ratio and then spotted onto filter paper. The samples were alkalinized (pH approximately 9.2) with potassium hydroxide at the time of assay and the compounds were extracted together with internal standard into di-isopropyl ether and then re-extracted into an aqueous phase with 0.1M phosphate buffer at pH 4. The chromatographic analysis was performed using an Agilent Technologies ChemStation LC System. The absorbance of the compounds was monitored at 333 nm. Mean extraction recoveries of AQ and DAQ were 49 and 48%, respectively. Intra-day and inter-day coefficients of variation were <10.5%. The limit of quantification was 50 nM for both compounds (sample size 100 microl). Both AQ and DAQ that were previously reported to be unstable have been stored on filter paper for at least 19 weeks. The method was applied on samples from healthy volunteers.     

Rapid and simple method to determine chloroquine and its desethylated metabolites in human microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the simultaneous determination of chloroquine (CQ) and its desethylated metabolites monodesethylchloroquine (DCQ) and bisdesethylchloroquine (BDCQ) in human liver microsomes. Analytes were separated on a C₁ column using methanol-water (70:30, v/v) and triethylamine (0.1% v/v) as the mobile phase. The fluorescence detector was set at 250 (excitation) and 380 nm (emission). Following protein precipitation with ice-cold acetonitrile, microsomal incubation supernatants were directly injected into the HPLC system. Typically, 200 μl of incubate were diluted with 200 μl of acetonitrile and 15 μl were injected. The limit of quantitation was 78 nM of CQ or metabolite. Intra-day variability averaged 2.9% for CQ, 1.5% for DCQ and 2.5% for BDCQ. Inter-day variability was 3.1% for CQ, 3.5% for DCQ and 3.7% for BDCQ. Mean accuracies were 100% for CQ and BDCQ and 102% for DCQ.     

Simple and cost-effective liquid chromatographic method for determination of pyrimethamine in whole blood samples dried on filter paper

A cost-effective HPLC method for determination of pyrimethamine (PYR) in human whole blood samples dried on filter paper (Whatman) is reported. Trimethoprim (TMP) was used as an internal standard. Whole blood spiked with PYR was transferred (100 microl) onto filter paper and dried at room temperature. Capillary blood samples (100 microl) after ingestion of three tablets of sulfadoxine-pyrimethamine (SP) by one subject were also tested. PYR and an internal standard (IS) TMP were extracted into di-isopropyl ether as bases and then re-extracted with 150 microl mobile phase. A C-18 column was used and the mobile phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid (750:300:2.5, v/v/v). The absorbances of PYR and IS were monitored at 270 nm. The limit of quantification was 40 ng/ml. The within- and between-assay coefficient of variations were <10% at the limit of quantification.     

Selective and sensitive liquid chromatographic assay of amodiaquine and desethylamodiaquine in whole blood spotted on filter paper

We have developed a sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection (340 nm) for the simultaneous quantification of amodiaquine (AQ) and its major metabolite, desethylamodiaquine (AQm) in a small volume (200 microl) of whole blood spotted on filter paper. The method involves liquid-liquid extraction with diethyl ether followed by elution from a reversed-phase phenyl column with an acidic (pH 2.8) mobile phase (25 mM KH2PO4-methanol; 80:20% (v/v) +1% (v/v) triethylamine). Calibration curves in spiked whole blood were linear from 100-2500 ng/ml (r2 > or = 0.99) for AQ and 200-2500 ng/ml (r2 > or = 0.99) for AQm. The limit of detection was 5 ng for AQ and 10 ng for AQm. The relative recovery at 150 ng/ml of AQ (n = 6) was 84.0% and at 300 ng/ml of AQm the relative recovery was 74.3%. The intra-assay coefficients of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 7.7, 8.9 and 6.2% (AQ) and 10.1, 5.4 and 3.9% (AQm), respectively. The inter-assay coefficient of variation at 150, 600 and 2250 ng/ml of AQ and 300, 600 and 2250 ng/ml of AQm were 5.2, 8.1 and 6.9% (AQ) and 3.3, 2.3 and 4.6% (AQm). There was no interference from other commonly used antimalarial and antipyretic drugs (chloroquine, quinine, sulfadoxine, pyrimethamine, artesunate, acetaminophen and salicylate). The method is particularly suitable for pharmacokinetic studies in settings where facilities for storing blood/plasma samples are not available.     

Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid-solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients > or = 0.997 (r2 > or = 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) < or = 3.8% for AQ-13 and its metabolites, and < or =2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 microl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 microl.     

Practical HPLC methods for the quantitative determination of common antimalarials in Africa

This article describes high-performance liquid chromatographic assays for the quantification of sulfadoxine (SDX), pyrimethamine (PYM), chloroquine (CQ), amodiaquine (AQ) and desethylamodiaquine (AQM) from whole blood. All four assays were set up and validated in Malawi using a common high-performance liquid chromatography platform and column and involved the use of simple mobile phase and extraction reagents. Calibration curves were linear (r(2)>0.95) in the ranges 5-100microg/ml, 50-1000, 150-1500, 100-1000 and 100-1000ng/ml for SDX, PYM, CQ, AQ and AQM, respectively. Intra-assay and inter-assay coefficients of variation were <15% at 3 points spanning the concentration range and <20% at the lower limit of quantification. The assays were specific with no interference from the other antimalarials described in this report. All four assays use liquid-liquid extraction, reversed-phase chromatography and UV detection and require between 50 and 200microl of blood. Because the assays share common instruments and reagents, they are cost-efficient and could be used to optimise antimalarial drug therapies in other resource poor settings.     

Simultaneous determination of a novel angiotensin II receptor blocking agent, losartan, and its metabolite in human plasma and urine by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the simultaneous determination of a new angiotensin II receptor blocking agent, losartan (DuP 753, MK-954, I), and its active metabolite, EXP3174 (II), in human plasma or urine is described. The two analytes and internal standard are extracted from plasma and urine at pH 2.5 by liquid—liquid extraction and analyzed on a cyano column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limit of quantification for both compounds in plasma is 5 ng/ml. The limit in urine is 20 and 10 ng/ml for I and II, respectively. The assay described has been successfully applied to samples from pharmacokinetic studies.     

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

A bioanalytical method for the determination of piperaquine in 100 microL blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Blood spots were cut into small pieces prior to addition of 0.3M perchloric acid, acetonitrile and phosphate buffer containing an internal standard. The liquid phase was loaded onto a mixed phase cation-exchange (MPC) solid-phase extraction column. Piperaquine and the internal standard were analysed by liquid chromatography and separated on a Chromolith Performance (100 mm x 4.6 mm) column with acetonitrile:phosphate buffer pH 2.5, I = 0.1 (8:92, v/v) at the flow of 3.5 mL/min. The UV detection was performed at 345 nm. The intra-assay precision was 12.0% at 0.150 microM, 7.3% at 1.25 microM and 7.3% at 2.25 microM. The inter-assay precision was 1.8% at 0.150 microM, 5.2% at 1.25 microM and 2.8% at 2.25 microM. The lower limit of quantification (LLOQ) was determined to 0.050 microM where the precision was 14.7%.     

Determination of lysergide in urine by high-performance liquid chromatography combined with electrospray ionisation mass spectrometry

A quantitative method which avoids derivatisation is described for the determination of lysergide (LSD) levels in urine. Sample preparation included addition of methysergide as an internal standard followed by solid-phase extraction. LSD was analysed on a system consisting of a C₁₈ stationary phase and a mobile phase of 0.1 M acetate buffer pH 8.0-acetonitrile-triethylamine (75:25:0.25, v/v). LSD was detected by electrospray ionisation mass spectrometry with selected ion monitoring. The quantification limit was 0.5 ng/ml and the method was linear up to 10 ng/ml of LSD in urine.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C₁₄ reversed-phase column (Zorbax Bonus-RP column) and a liquid–liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid–liquid extraction. The mobile phase consists of acetonitrile–0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone ®) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C₁₈, 250x4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m² of mitoxantrone by intravenous infusion for 10 min.     

Bioanalysis and pharmacokinetics of the p38 MAPkinase inhibitor SB202190 in rats

We have developed a sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for the quantification of the p38 MAPkinase inhibitor SB202190 in serum, kidney homogenates and urine samples. Liquid-liquid extraction of SB202190 from the samples was performed using diethylether after adding a derivative of SB202190 as internal standard (I.S.). Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of acetonitrile-water-trifluoroacetic acid (30:70:0.1, v/v/v; pH 2.0). Both drug and I.S. were measured at 350 nm and eluted at 5.0 and 10.6 min, respectively. Peak-height ratios of the drug and the I.S. were used for the quantification of SB202190 from the different matrixes. The limit of quantitation of SB202190 in serum, kidney and urine were 0.25 microg/ml, 1 microg/g and 1 microg/ml, respectively. The average recoveries were 74, 75 and 92% in serum, kidney and urine, respectively. The intra- and inter-day precision (% CV) and accuracy (% bias) were below 15% for all concentrations. The method was successfully applied for a pharmacokinetic study of SB202190 in rats.     

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.     

Measurement of blood coagulation Factor XIIIa formation in plasma containing glycyl-L-prolyl-L-arginyl-L-proline

A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Control of propranolol intake by direct chromatographic detection of alpha-naphthoxylactic acid in urine

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.     

Simple determination of cyclosporine in human whole blood by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of cyclosporine (CyA, also known as cyclosporin A) in human whole blood. The method entailed direct injection of the blood samples after deproteination using acetonitrile. Chromatography was carried out using an ODS column under isocratic elution with acetonitrile-5mM disodium hydrogen phosphate (75:25, v/v), pH 5.1 at 70 degrees C and a detector set at 210 nm. The mean absolute recovery of cyclosporine from blood was 97%, and the linearity was assessed in the range of 100-3000 ng/ml blood, with a correlation coefficient of greater than 0.999. The limit of quantification and detection of the present method were 100 and 50 ng/ml, respectively. This method has been used to analyze several hundred human blood samples for bioavailability studies.     

Simultaneous determination of levofloxacin, gatifloxacin and moxifloxacin in serum by liquid chromatography with column switching

Liquid chromatography with a column-switching technique was developed for simultaneous direct quantification of levofloxacin, gatifloxacin and moxifloxacin in human serum. Serum samples were injected on a LiChroCART 4-4 pre-column (PC) filled with a LiChrospher 100 RP-18, 5 microm where fluoroquinolones (FQs) were purified and concentrated. The FQs were back-flushed from the PC and then separated on a Supelcosil ABZ+ Plus (150 mm x 4.6 mm i.d.) analytical column with a mobile phase containing 10 mM phosphate buffer (pH 2.5), acetonitrile (88:12, v/v) and 2mM tetrabutyl ammonium bromide. The effects of ion-pair reagents, buffer type, pH and acetonitrile concentrations in the mobile phase on the separation of the three FQs were investigated. Fluorescence detection provided sufficient sensitivity to achieve a quantification limit of 125 ng/ml for levofloxacin and moxifloxacin; 162.5 ng/ml for gatifloxacin with a 5 microl sample size. The on-line process of extraction avoids time-consuming treatment of the samples before injection and run time is shortened. The recovery, selectivity, linearity, precision and accuracy of the method are convenient for pharmacokinetic studies or routine assays.     

Simple liquid chromatographic method for the determination of cefotaxime in human and rat plasma

A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.