Journal of Chromatography B (v.859, #1)

Affinity chromatography using biocompatible and reusable biotinylated membranes by S. Govender; E.P. Jacobs; M.W. Bredenkamp; P. Swart (1-8).
A novel, reusable biotinylated affinity chromatography strategy for the bio-specific binding of bioactive avidin tagged enzymes or polypeptides is reported. Using an avidin coupled peroxidase fusion protein as a test system; non-specific protein shielding and matrix regeneration were also shown. The amphiphilic surfactant Pluronic® F108 was used as an affinity linker, by non-covalent binding to membrane chromatographic matrices while the terminal hydroxyl groups of Pluronic were covalently coupled to the biological ligand biotin. Planar nonporous membranes of varying surface chemistry were synthesised to test the matrix dependent affinity binding of biotinylated Pluronic and their respective ability to resist non-specific protein adsorption. Membrane regeneration using sodium dodecyl sulphate (SDS) was capable of displacing both adsorbed proteins and Pluronic. SDS micelles (34 mM) were effective in desorbing membrane bound protein while 5 mM SDS removed up to 85% of the bound ligand after 20 h incubation at 20 °C. In this study, polyvinylidene membranes had the highest ligand binding capacity of 0.22 mg cm−2 and specific, competitive affinity binding of avidin-peroxidase was shown in the presence of up to 0.2 mg ml−1 ‘contaminant’ proteins. The resultant biocompatible affinity chromatographic system was regenerated and reused with no significant change in performance for up to five cycles.
Keywords: Affinity chromatography; Avidin tagged proteins; Biotinylation; Pluronic; Regeneration;

Development and application of procedures for the highly sensitive quantification of cyclosarin enantiomers in hemolysed swine blood samples by Georg Reiter; Marianne Koller; Horst Thiermann; Frederic Dorandeu; John Mikler; Franz Worek (9-15).
The present study was initiated to develop a sensitive method for the analysis of cyclosarin (O-cyclohexyl methylphosphonofluoridate, GF) enantiomers in biological samples utilizing classical configurations of GC–MS and automated solid phase extraction. To achieve this goal, a specific procedure had to be developed to extract cyclosarin from swine blood samples thereby stabilising and minimising the racemisation/deracemisation of its enantiomers. The chiral stationary phase was GAMMA DEX™ (gamma cyclodextrin), on which GF and deuterated GF enantiomers were baseline-resolved. The limit of detection was 1 pg for (−)-GF with GC–EI-MS and 5 pg for (+)-GF with GC–NCI-MS. The absolute recovery of the overall procedure for sample preparation was 85%. After an intravenous infusion of a supralethal dose of GF in anaesthetised swine only (−)-GF could be quantified, (+)-GF was not detected.
Keywords: Gas chromatography–mass spectrometry; O-Cyclohexyl methylphosphonofluoridate; Cyclosarin; GF; Enantiomers; Solid phase extraction; Cyclodextrin; Poisoning; Nerve agents; Toxicokinetics; Swine; Oxime; Reactivator; Acetylcholinesterase;

Streamline Direct HST is a new kind of mixed-mode adsorbent with cation exchange ligand, especially developed for the expanded bed adsorption process, which can capture target protein directly from the moderate ionic strength feedstock without the need of dilution or other additives. In this study, the isotherm adsorption behaviors and the isocratic retention factors of bovine serum albumin (BSA) on Streamline Direct HST were measured, and the corresponding adsorption mechanisms were also described. The results indicated that Streamline Direct HST shows the typical property of salt-independent adsorption and the maximum binding capacity of BSA occurs near the isoelectric point of BSA. When there are some amounts of electrostatic repulsion protein–adsorbent interactions, the multilayer adsorption could be found, and high salt concentration does not favor the adsorption of protein. A patch-controlled adsorption process and an oriented adsorption model are proposed for describing the adsorption behaviors under electrostatic repulsion condition.
Keywords: Mixed-mode chromatography; Streamline Direct HST; Multilayer adsorption; Patch-controlled adsorption; Oriented adsorption;

Sensitive and rapid liquid chromatography/tandem mass spectrometric assay for the quantification of piperaquine in human plasma by Puran Singhal; Ashwani Gaur; Anirudh Gautam; Brijesh Varshney; Jyoti Paliwal; Vijay Batra (24-29).
A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC–MS/MS) method was developed and validated for quantification of piperaquine, an antimalarial drug, in human plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method involved a simple protein precipitation with methanol followed by rapid isocratic elution of analytes with 10 mM ammonium acetate buffer/methanol/formic acid/ammonia solution (25/75/0.2/0.15, v/v) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and quantification by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 535.3 → 288.2 and m/z 409.1 → 205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 1.0–250.2 ng/mL for piperaquine in plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) in plasma were 0.2 and 1.0 ng/mL, respectively. Acceptable precision and accuracy (±20% deviation for LLOQ standard and ±15% deviation for other standards from the respective nominal concentration) were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully applied to analyze human plasma samples from phase-1 clinical studies. The mean pharmacokinetic parameters of piperaquine following 1000 mg oral dose: observed maximum plasma concentration (C max), time to maximum plasma concentration (T max) and elimination half-life (T 1/2) were 46.1 ng/mL, 3.8 h and 13 days, respectively.
Keywords: Antimalarial: Piperaquine; Liquid chromatography/tandem mass spectrometry; Human plasma;

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5 mM NDA, 40 mM NaCN and 20 mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0 mM β-cyclodextrin (β-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 × 10−9  M, 5.6 × 10−9  M and 7.2 × 10−9  M, respectively (S/N  = 3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With β-casein as the analysis protein, this method was successfully validated.
Keywords: Phosphoamino acids; β-Cyclodextrin; Capillary electrophoresis; Naphthalene-2,3-dicarboxaldehyde;

Phosphodiesterases are drug targets for treating various diseases. Inhibition of these can increase cAMP and cGMP levels, which can affect a variety of physiological responses. Here we report a new method for determining PDE activity by combining high-performance liquid chromatography and tandem mass spectrometry. Characteristic peaks of the substrates, cGMP or cAMP and products, GMP or AMP, were identified in positive-ion electrospray ionization using multiple reaction monitoring. The method can be applied to determine activity of PDE inhibitors. Our results showed that this new method was fast, sensitive and highly reproducible.
Keywords: Phosphodiesterases; Mass spectrometry; Sildenafil; cGMP; cAMP;

Chronic combination immunosuppressive regimens are commonly prescribed to renal transplant recipients. To develop an assay method for pharmacokinetic studies and therapeutic drug monitoring of multiple immunosuppressives, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) approach for the simultaneous analysis of several glucocorticoids, mycophenolic acid (MPA) and mycophenolic acid glucuronide (MPAG) was investigated. The resultant method utilized a gradient reverse phase separation over a Symmetry C18 column using an ammonium acetate–methanol mobile phase at pH 3.5. The analytes were detected by coupling the chromatography system via electrospray to a triple quadrupole mass spectrometer. Multiple-reaction monitoring in the negative mode ion (MH-/product) was employed selecting MPA at 319.1/190.9, MPAG at 495.1/191.0, dexamethasone at 391.0/361.0, hydrocortisone at 361.1/331.1, methylprednisolone at 373.1/343.1, prednisone at 357.1/327.2, and prednisolone at 359.1/329.1. The calibration curve concentrations ranged from 3.60 ng/mL to 50 μg/mL with the lowest limit of quantitation for corticosteroids being 3.60–7.20 ng/mL and 0.656–6.75 μg/mL for MPA and MPAG, respectively. The relative standard deviation for quality control intraday variation and interday variation was between 0.76% and 9.57% for all analytes. This assay offers a versatile, unique method for multi-analyte immunosuppressive determinations during combination immunosuppression.
Keywords: Immunosuppressives; Mycophenolic acid; Glucocorticoids; Assay; LC/MS/MS;

A simple, sensitive and reliable method was developed to determine simultaneously the concentrations of thienorphine and its metabolite thienorphine glucuronide conjugate in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The metabolite was identified by MS: thienorphine glucuronide conjugate. Sample preparation involved protein precipitation with methanol. Analytes were separated on Finnigan BetaBasic-18 column (150 mm × 2.1 mm i.d., 5 μm) using methanol: water: formic acid (56:44:0.1, v/v/v) as mobile phase at a flow rate of 0.2 ml/min. The method had a linear calibration curve over the concentration range of 0.1–50 ng/ml for thienorphine and 2–1000 ng/ml for thienorphine glucuronide conjugate, respectively. LOQ of thienorphine and thienorphine glucuronide conjugate was 0.1 and 2 ng/ml, respectively. The intra- and inter-batch precisions were less than 12% and their recoveries were greater than 80%. Pharmacokinetic data of thienorphine and its metabolite thienorphine glucuronide conjugate obtained with this method following a single oral dose of 3 mg/kg thienorphine to rats were also reported for the first time.
Keywords: Thienorphine; Thienorphine glucuronide conjugate; LC–MS/MS; Rat plasma; Pharmacokinetics;

A liquid chromatography–mass spectrometry (LC–MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N 1-acetyltriethylenetetramine (MAT) and N 1,N 10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC–MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 MΩ), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M + H]+ values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N 1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.
Keywords: LC–MS; Triethylenetetramine; Metabolite; Copper chelator; Diabetic heart failure;

A simple and sensitive high performance liquid chromatography method using UV detection (HPLC-UV) for the determination of meloxicam in human plasma was developed and validated. After extraction with diethyl ether, the chromatographic separation of meloxicam was carried out using a reverse phase Sunfire C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile–20 mM potassium hydrogen phosphate (40:60, v/v, pH 3.5) and UV detection at a wavelength of 355 nm. The flow rate of mobile phase was 1.2 ml/min and the retention time of meloxicam and internal standard, piroxicam, was found to be 11.6 and 6.3 min, respectively. The calibration curve was linear within the concentration range, 10–2400 ng/ml (r 2  > 0.9999). The lower limit of quantification was 10 ng/ml. This method improved the sensitivity for the quantification of meloxicam in plasma using a HPLC-UV. The mean accuracy was 98–114%. The coefficient of variation (precision) in the intra- and inter-day validation was 1.6–4.3 and 2.4–7.3%, respectively. The pharmacokinetics of meloxicam was evaluated after administering an oral dose of 15 mg to 11 healthy Korean subjects. The AUCinf, C max, t max and t 1/2 were 42.4 ± 13.2 μg h/ml, 1445.7 ± 305.5 ng/ml, 4.1 ± 0.3 h and 22.0 ± 4.9 h, respectively.
Keywords: Meloxicam; HPLC; Pharmacokinetics;

A bioanalytical method for the analysis of oseltamivir (OP) and its metabolite oseltamivir carboxylate (OC) in human plasma, saliva and urine using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. OP and OC were analysed on a ZIC-HILIC column (50 mm × 2.1 mm) using a mobile phase gradient containing acetonitrile–ammonium acetate buffer (pH 3.5; 10 mM) at a flow rate of 500 μL/min. The method was validated according to published FDA guidelines and showed excellent performance. The lower limit of quantification for OP was determined to be 1, 1 and 5 ng/mL for plasma, saliva and urine, respectively and for OC was 10, 10 and 30 ng/mL for plasma, saliva and urine, respectively. The upper limit of quantification for OP was determined to be 600, 300 and 1500 ng/mL for plasma, saliva and urine, respectively and for OC was 10,000, 10,000 and 30,000 ng/mL for plasma, saliva and urine, respectively. The within-day and between-day precisions expressed as R.S.D., were lower than 5% at all tested concentrations for all matrices and below 12% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. No matrix effects were detected for OP or OC in plasma or saliva. Residues from the urine matrix (most likely salts) caused some ion suppression for both OP and its deuterated internal standard but had no effect on OC or its deuterated internal standard. The suppression did not affect the quantification of OP.
Keywords: Avian influenza; Birdflu; High throughput; Ion suppression; Liquid chromatography/tandem mass spectrometry (LC/MS/MS); Oseltamivir; Stable isotope labeled (SIL) internal standard; Tamiflu;

Matrix effects resulting in ion suppression or enhancement have been shown to be a source of variability and inaccuracy in bioanalytical mass spectrometry. Glycerophosphocholines may cause significant matrix ionization effects during quantitative LC/MS/MS analysis and are known to fragment to form characteristic ions (m/z 184) in electrospray mass spectrometry. This ion was used to monitor ion suppression effects in the determination of hydrocodone and pseudoephedrine in human plasma as a means to track and avoid these effects. The m/z 184 ion fragment was detected in both plasma extracts and solutions of phosphatidylcholine. Post-column infusion studies showed that the ion suppression for both drugs and internal standards correlated with the elution of phospholipids. HPLC conditions were adjusted to chromatographically resolve the peaks of interest from the phospholipids. Upon repeated injection, the elution time of the phospholipids decreased while elution of the analyte peaks remained unchanged. This resulted in co-elution and significantly affected peak shape and internal standard response for the analytes. It was decided to use the phospholipid fragment to monitor this matrix effect in validation samples. The resulting method demonstrated intra-day and inter-day precision within 4.5 and 5.6% for hydrocodone and pseudoephedrine, respectively, and accuracy within 8.9 and 8.7% for hydrocodone, and pseudoephedrine, respectively. There was no statistically significant difference in the internal standard response for the determination with and without monitoring the phospholipid fragment ion. We found that monitoring the phospholipid fragment was useful in method development to avoid the matrix effects, and in routine analysis to provide a practical way to ensure the avoidance of matrix effects in each individual sample.
Keywords: Matrix effects; LC/MS/MS; HPLC; Validation;

We developed and validated an HPLC method for determination of 1-chloro-2,4-dinitrobenzene (CDNB) and its glutathione conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) to study the kinetics and mechanisms involved in DNP-SG formation and efflux, as a probe for human placental metabolism and transport. This method combines use of 3 μm solid phase, rapid mobile phase gradient with dual wavelength ultraviolet detection to permit determination of a lipophilic parent compound and its hydrophilic metabolites in a single short run. The selectivity, linearity, accuracy, precision, relative recovery and stability of the assay are sufficient for determining CDNB, DNP-SG and its metabolites from buffer and tissue samples to support placental drug metabolism and transport studies.
Keywords: 1-Chloro-2,4-dinitrobenzene (CDNB); 2,4-Dinitrophenyl-S-glutathione (DNP-SG); Placental metabolism and transport; GSTP1-1; ABCC2; ABCG2;

A sensitive assay for determination of rifalazil (also known as ABI-1648 and KRM-1648) in human plasma is described. The analytical method utilizes liquid–liquid extraction of plasma with methyl tert-butyl ether, followed by reversed-phase liquid chromatography with a C18 column and a mobile phase gradient utilizing 0.1% formic acid in water and acetonitrile, respectively. Electrospray mass spectrometry in the positive ion mode with selected reaction monitoring of rifalazil and an isotope labeled internal standard, 13C4-rifalazil (ABI-9901) was used for selective and sensitive detection. The calibration range was 0.050–50 ng/mL plasma using 200 μL plasma sample volume. The absolute extraction recovery of rifalazil from K2-EDTA plasma, evaluated at three concentration levels, was 88.6–97.3%, and the recovery for the internal standard was 96.8%. A study of plasma matrix effects showed a peak area response at 90–99% compared to neat solutions for both rifalazil and the internal standard. Stability evaluation of rifalazil in plasma, whole blood and methanol showed that the analyte stability was adequate when stored under study conditions. The precision, as evaluated in three validation batches, was consistent for fortified plasma quality control (QC) samples at four concentration levels, with ≤6% R.S.D. except for at the lowest quality control level where it was 10.7% R.S.D. The accuracy for QC samples (difference between found and nominal concentration) ranged from −2.3% to 5.1%. Similar precision and accuracy values were obtained over 6 months of routine application of this method. It was concluded that the performance improved markedly during routine operation by replacing a closely related structural analog internal standard with the stable isotope internal standard.
Keywords: Rifalazil; ABI-1648; KRM-1648; Human plasma; Isotope labeled internal standard; Quantification; Electrospray;

Affinity purification of serine proteinase from Deinagkistrodon acutus venom by Yu Xin; Dexian Dong; Ting Wang; Rongxiu Li (111-118).
An affinity protocol was developed for the preparation of the main serine proteinase from Deinagkistrodon acutus venom on industrial scales. As affinity ligand, l-arginine was composed to medium and its structure was confirmed by ESI-MS analysis. The purification process consisted of one major affinity chromatography step to remove more than 95% of other proteins, and a polishing step of DEAE ion-exchange chromatography for removal of minor contaminants. The serine proteinase was 100% pure analyzed on HPLC Vydac C4 column, 99.4% on TSK G3000SW column, and 97.7% with SDS-PAGE analysis. The yield of the main serine proteinase was 3.6% of crude venom protein, the recoveries of typical fibrinogen (Fg) clotting activity and arginine esterase activity of serine proteinase were 82.2% and 84%, higher than those of other reported traditional protocols, the proteinase also showed arginine amidase activity. Reducing SDS-PAGE analysis showed that the arginine esterase was a single polypeptide with the mass of ∼40 kDa, while MALDI-TOF-TOF-MS analysis showed that the purified proteinase should be a ∼34 kDa glycoprotein. The desorption constant K d and the theoretical maximum absorption Q max on the affinity medium were 9.93 × 10−5 and 38.1 mg/g medium in absorption analysis.
Keywords: Affinity chromatography; Serine proteinases; Deinagkistrodon acutus venom;

Although Laggera pterodonta, a folk medicine has been widely used for several centuries as an antiviral agent, there have been no studies to identify its antiviral components. A bioassay-guided phytochemical examination of L. pterodonta disclosed that its aqueous extract, which was made up of three dicaffeoylquinic acids showed significant inhibitory activity of virus replication. Then a simple and efficient preparative high-speed counter-current chromatography (HSCCC) method was successfully established by using ethyl acetate–n-butanol–water (3:2:5, v/v) as the two-phase solvent system to isolate and purify three bioactive dicaffeoylquinic acids, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid. The flow rate was 1.5 ml/min and revolution speed was 800 rpm. The isolation was done in less than 6 h, and 34.6 mg of 3,5-O-dicaffeoylquinic acid, 29.4 mg of 3,4-O-dicaffeoylquinic acid and 27.1 mg of 4,5-O-dicaffeoylquinic acid was yielded from 600 mg of the crude sample in one-step separation with the purity of 98.3%, 96.7% and 96.2%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of these three bioactive dicaffeoylquinic acids were identified by ultraviolet (UV), electrospray ionization mass spectrometry (ESI-MS), proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). In the antiviral experiment, three dicaffeoylquinic acids all showed significant inhibitory activity against herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) and influenza viruses A (IVA) in vitro with high selectivity indexes. However, among the three compounds, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid were the more active than 3,4-O-dicaffeoylquinic acid against HSV-1, HSV-2 and IVA. This study demonstrated a direct link between the antiviral activity of L. pterodonta and the three dicaffeoylquinic acids.
Keywords: Laggera pterodonta; High-speed counter-current chromatography; 3,5-O-Dicaffeoylquinic acid; 3,4-O-Dicaffeoylquinic acid; 4,5-O-Dicaffeoylquinic acid; Antiviral activity;

A novel determination method for josamycin (JOS) based on capillary electrophoresis–electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 μm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 μm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3 2+ and 50 mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8 s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 μg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Josamycin; Tris(2,2′-bipyridyl) ruthenium(II); Plasma;

High-performance liquid chromatography method for quantifying sphingomyelin in rat brain by Diana J. Azzam; Julnar A. Usta; Youssef Mouneimne; Jimmy A. El Hokayem; Mohamad A. Mikati (131-136).
A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile–methanol–water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025–0.4 mg/ml sphingomyelin (R 2  > 0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.
Keywords: HPLC; Sphingomyelin; Brain; Internal standard; Rat; Isocratic; Quantification;

A field-adapted HPLC method for determination of amodiaquine and its metabolite in whole blood dried on filter paper by M. Ntale; M. Mahindi; J.W. Ogwal-Okeng; L.L. Gustafsson; O. Beck (137-140).
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 ≈ 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 0.1 M 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 μl). 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.
Keywords: Amodiaquine; Desethylamodiaquine; Filter paper;

A simple and improved HPLC method with fluorometric detection for simultaneous determination of dextromethorphan (DM) and its three metabolites (dextrorphan (DX), 3-methoxymorphinan (MM), 3-hydroxymorphinan (HM)) in human plasma was developed and validated. The method involved a simple and efficient extraction protocol using an n-heptane/ethyl acetate (1:1) solvent mixture that achieved recoveries of 70–90% with an insignificant interference from the plasma matrix. The analysis was performed on a phenyl column with isocratic elution, a mobile phase composed of 20% methanol, 30% acetonitrile, and 50% KH2PO4 buffer (10 mM, with adding 0.02% of TEA; adjusted with phosphoric acid to pH 3.5), and a run time of only 15 min. Linear calibration curves were constructed in the concentration range of 1–200 nM for DM and its three metabolites. The lower limit of quantitation (LLOQ) in human plasma was 1 nM for each compound. The coefficient of variation and RSE% of the intraday and interday analyses for DM and its three metabolites all complied with USFDA requirements. This analytical method was preliminarily applied to determine the polymorphic functions of CYP2D6 and CYP3A4 in the metabolic pathway of DM to DX and then to HM.
Keywords: Dextromethorphan; Dextrorphan; Methoxymorphinan; Hydroxymorphinan; Phenyl column;