Innovative HPTLC method for simultaneous determination of ternary mixture of certain DMARDs in real samples of rheumatoid arthritis patients: an application of quality by design approach.

A uniquely developed high performance thin-layer chromatographic (HPTLC) method coupled with UV detection was applied using quality by design approach (QbD) for simultaneous determination of methotrexate (MTX), sulfasalazine (SSZ) and hydroxychloroquine (HCQ) in serum and urine samples of rheumatoid arthritis patients. MTX, SSZ with HCQ are the most common disease-modifying antirheumatic drugs (DMARDs) combination used for the treatment of rheumatoid arthritis. This ternary mixture with montelukast (MK) added as internal standard, were separated by using a mixture of ethyl acetate: methanol: 25% ammonia, (8: 2: 3, v/v/v) as a mobile phase system. The separation was achieved on HPTLC precoated silica gel plate 60 F254 and the detection was carried out at 306 nm for MTX and at 340 nm for both SSZ and HCQ. The design was planned to obtain the most optimum retardation factors (Rf) with best resolution. The Rf values for MTX, SSZ, MK and HCQ were of 0.31 ±â€¯0.03, 0.62 ±â€¯0.02, 0.71 ±â€¯0.02 and 0.83 ±â€¯0.03; respectively. The interactive response optimizer achieved the most favorable conditions with acceptable composite desirability of 0.9703. Linear relationship with good correlation coefficients (r = 0.9990-0.9994) were also obtained with detection and quantification limits of 13.94-260.64 and 46.84-1810.01(ng/ml); respectively. The suggested method was established in accordance with the guidelines of Food and Drug Administration (FDA). The established QbD-HPTLC method achieved simple, sensitive and selective quantification of the studied drugs in serum and urine samples in the presence of their metabolites with no interferences. This method can be extended effectively for therapeutic drug monitoring and pharmacokinetics studies of these drugs.

Cross-Reactivity of Chloroquine and Hydroxychloroquine With DRI Amphetamine Immunoassay.

BACKGROUND: Chloroquine and hydroxychloroquine are medical drugs used to treat the chemoprophylaxis of malaria and a second-line anti-inflammatory drug. METHODS: We performed a study of cross-reactivity of chloroquine and hydroxychloroquine in the DRI Amphetamine Assay inspired by a case report of a self-ingestion of chloroquine after a family dispute, that involved the following: (1) an in vitro study with control samples of healthy subjects, (2) an in vivo study with samples of patients with rheumatoid arthritis, and (3) an evaluation of the cross-reactivity of chloroquine and hydroxychloroquine in 3 additional immunoassays. RESULTS: In the case report, the Amphetamine DRI assay resulted positive both at 1000 ng/mL cutoff (1507 and 1137 ng/mL) and at 500 ng/mL cutoff (1178 and 642 ng/mL). Chloroquine urine levels were 103,900 and 100,900 ng/mL at 5 and 9 hours after ingestion. The results with control samples showed a positive cross-reactivity of chloroquine in the DRI Amphetamine Assay (approximately 0.74% and 0.89% at cutoff of 1000 and 500 ng/mL, respectively). Hydroxychloroquine did not cross-react with the DRI Amphetamine Assay up to 1,000,000 ng/mL. In patients treated with chloroquine or hydroxychloroquine, DRI Amphetamine did not produce false-positive results. The comparative assay study showed a positive cross-reactivity of chloroquine in the Emit II Plus Amphetamines Assay with control samples. CONCLUSIONS: Chloroquine can cause false-positive results in the DRI Amphetamine Assay when it is present at high concentrations. Hydroxychloroquine did not produce false-positive results neither in the DRI Amphetamine Assay nor in the others immunoassays evaluated.

Stereoselective determination of hydroxychloro-quine and its major metabolites in human urine by solid-phase microextraction and HPLC.

The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid-phase microextraction. The chromatographic separation was performed on a Chiralcel OD-H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25 degrees C and the desorption time was 3 min using methanol (100%). PDMS-DVB 60 microm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50-1000 ng/mL for HCQ enantiomers and over the range of 42-416 ng/mL for DCQ and DHCQ enantiomers. Within-day and between-day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)-(S)-enantiomer excreted was higher than its antipode.

Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase microextraction and CE.

Liquid-phase microextraction based on polypropylene hollow fibers and CE were applied for the chiral determination of hydroxychloroquine (HCQ) and its metabolites (desethylchloroquine, DCQ; desethylhydroxychloroquine, DHCQ; bisdesethylchloroquine, BDCQ) in human urine. The analytes were extracted from 3 mL of urine spiked with the internal standard (metoprolol) and alkalinized with 250 muL of 2 M NaOH. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the hollow fiber. The electrophoretic separations were carried out in 100 mmol/L Tris buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% w/v S-beta-CD and 30 mg/mL HP-beta-CD with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng/mL for each HCQ stereoisomer and 21-333 ng/mL for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels for each stereoisomer and were lower than 15%. The developed method was applied for the determination of the cumulative urinary excretion of HCQ, DCQ, and DHCQ after oral administration of rac-HCQ to a health volunteer. The results obtained are in agreement with previous literature data.

Enantioselective disposition of hydroxychloroquine after a single oral dose of the racemate to healthy subjects.

1. Stereoselectivity in the disposition of hydroxychloroquine was investigated in 23 healthy males following a single oral dose of 200 mg racemic HCQ (rac-HCQ) sulphate. Total concentrations (R+S) and R/S ratios of HCQ and its metabolites were measured by stereoselective h.p.l.c. 2. HCQ was detected in whole blood and urine, up to 91 and 85 days after dosing, respectively. Metabolites could not be detected in whole blood while in urine detectable concentrations were still present after 85 days. The blood concentrations of HCQ enantiomers were measurable until 168 h post-dose. 3. R(-)-HCQ accounted for 62 +/- 3% (mean +/- s.d.) of the AUC of rac-HCQ AUC. The elimination half-life of S(+)-HCQ (457 +/- 122 h) was significantly shorter than that of R(-)-HCQ (526 +/- 140 h), partly due to its faster urinary excretion and hepatic metabolism. Its renal clearance was twice that of R(-)-HCQ (4.61 +/- 4.01 vs 1.79 +/- 1.30 1 h-1), and metabolites derived from the S-isomer represented 80-90% of the urinary recovery of the dose. 4. Over 85 days, 4.4 +/- 2.9 and 3.3 +/- 1.8% of the dose was recovered in urine as unchanged S(+)-HCQ and R(-)-HCQ, respectively. For the first 2 weeks, S(+)-HCQ excretion rate clearly surpassed that of R(-)-HCQ whereas afterwards the inverse was observed. However, since the first 2 weeks account for 95% of rac-HCQ renal excretion, the total urinary excretion of S(+)-HCQ clearly surpassed that of R(-)-HCQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the stereoisomers of hydroxychloroquine and its major metabolites in plasma and urine following a single oral administration of racemic hydroxychloroquine.

Plaquenil (hydroxychloroquine, HCQ; Sanofi Winthrop Pharmaceuticals, New York, NY) is a stereoisomeric drug administered as a racemic (50: 50) mixture of two isomeric forms--(+) and (-) HCQ. To correlate clinical efficiency accurately with dose, it is necessary to determine the fate of both isomers. This will allow for the optimization of clinical dosing. A method has been developed for the quantitation of (+) and (-) HCQ and its major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ), in plasma and in urine. Application of this method in a preliminary study in four human volunteers is reported. After a single oral dose of 200 mg of Plaquenil, the average enantiomeric ratio of (+) to (-) HCQ was approximately 1 over an 8-hour period. However, the average cumulative 48-hour excretion of HCQ, DHCQ, and DCQ showed stereoselective excretion.

Enantioselective determination of hydroxychloroquine and its major metabolites in urine and the observation of a reversal in the (+)/(-)-hydroxychloroquine ratio.

A sequential achiral-chiral high-performance liquid chromatographic system has been developed for the quantitation in urine of the enantiomers of hydroxychloroquine (HCQ), and of its 3 major metabolites, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ). HCQ and its metabolites were separated and quantified on a cyano-bonded phase, and the enantiomeric ratios were determined using a Chiral-AGP chiral stationary phase. The assay validation and application of this method to a preliminary study in a human volunteer are presented. In this subject, the initial 0-4 h urine contained the 2 HCQ enantiomers in a ratio of (+)-HCQ:(-)-HCQ of 3:2; by the 2,064 h of the study, this ratio had reversed to (+)-HCQ:(-)-HCQ of 3:7.

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.

Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method using fluorescence detection is described for the quantification of hydroxychloroquine (HCQ) and three of its metabolites in blood and urine samples. The method is selective, permitting quantification of analytes without interferences from chloroquine or quinine in the sample. Detection limits for HCQ, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylchloroquine are 10, 30, 5, and 5 ppb, respectively, for a 100-microliters blood or urine sample. The internally standardized method requires only one extraction step and utilizes normal-phase HPLC conditions including an amine modifier in the mobile phase. These conditions facilitate fluorescence detection, selective separation, and acceptable peak shapes. A mobile phase of 0.5% n-butylamine in methanol-hexane-methyl tert. butyl ether (1:1:1) is used in the analysis. Analysis of blood and urine samples from two healthy volunteers given 400 mg of Plaquenil (310 mg of HCQ base) weekly for four weeks provided data on HCQ metabolism for the two persons during the recommended chemoprophylactic regimen for malaria.