Hassan G. Fouda

Identification of potentially neurotoxic pyridinium metabolite in the urine of schizophrenic patients treated with haloperidol

Evidence that the parkinsonian inducing agent MPTP is biotransformed to a pyridinium species that selectively destroys nigrostriatal neurons in humans and subhuman primates has prompted studies to evaluate the metabolic fate of the structurally related neuroleptic agent haloperidol. With the aid of a highly sophisticated atmospheric pressure ionspray HPLC/MS/MS assay, unambiguous evidence has been obtained for the presence of the haloperidol pyridinium species in extracts of urine obtained from haloperidol-treated patients and in extracts of NADPH-supplemented human liver microsomal incubation mixtures containing haloperidol. The potential significance of the formation of this putative neurotoxic pyridinium species is considered.

Automated sample preparation using membrane microtiter extraction for bioanalytical mass spectrometry.

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.

Quantitative analysis by high-performance liquid chromatography atmospheric pressure chemical ionization mass spectrometry: The determination of the renin inhibitor CP-80,794 in human serum.

A specific method was developed for the quantitative determination of the renin inhibitor CP-BO,794. Serum extracts containing the drug and an internal standard were injected into a standard reverse-phase high-performance liquid chromatography (HPLC) column. The mobile phase, methanol/water (8/2), flowed at 1 ml/min through the column and then via a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source. The assay minimum limit of quantification was 50 pg/mL. It exhibits satisfactory accuracy and precision over the range 50 pg/ml, to 10 ng/mL. A minor modification of the HPLC mobile phase was necessary to attain extremely low detection limits. The addition of a structural analogue contributed to enhancing the precision of the assay.

Mass spectral characterization of tetracyclines by electrospray ionization, H/D exchange, and multiple stage mass spectrometry

Electrospray ionization (ESI) and collisionally induced dissociation (CID) mass spectra were obtained for five tetracyclines and the corresponding compounds in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The number of labile hydrogens, x, could easily be determined from a comparison of ESI spectra obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M + H]+ and [M – H]− ions and the exchanged analogs, [M(Dx) + D]+ and [M(Dx) − D]−, and produced by ESI using a Sciex API-IIIplus and a Finnigan LCQ ion trap mass spectrometer. Compositions of product ions and mechanisms of decomposition were determined by comparison of the MSN spectra of the un-deuterated and deuterated species. Protonated tetracyclines dissociate initially by loss of H2O (D2O) and NH3 (ND3) if there is a tertiary OH at C-6. The loss of H2O (D2O) is the lower energy process. Tetracyclines without the tertiary OH at C-6 lose only NH3 (ND3) initially. MSN experiments showed easily understandable losses of HDO, HN(CH3)2, CH3 - N=CH2, and CO from fragment ions. The major fragment ions do not come from cleavage reactions of the species protonated at the most basic site. Deprotonated tetracyclines had similar CID spectra, with less fragmentation than those observed for the protonated tetracyclines. The lowest energy decomposition paths for the deprotonated tetracyclines are the competitive loss of NH3 (ND3) or HNCO (DNCO). Product ions appear to be formed by charge remote decompositions of species de-protonated at the C-10 phenol.

Quantitative determination of the antibiotic azithromycin in human serum by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization mass spectrometry: correlation with a standard HPLC-electrochemical method.

A specific assay for the quantitative determination of the new antibiotic azithromycin in a low volume of human serum is described. The assay uses on-line high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI). Deuterium-labeled azithromycin was synthesized and used as the internal standard of the assay. The drug and the internal standard are extracted from 50 μl of serum, and aliquots are injected onto a standard reverse-phase HPLC column. The effluent from the HPLC column at 1 ml/min is introduced into the atmospheric pressure source of a SCIEX API III mass spectrometer. Azithromycin concentrations in serum are determined by the selected monitoring of the protonated molecular ions of the drug and the internal standard. Our assay yields accurate and precise results over the range 10 ng/ml to 250 ng/ml. The correlation between the assay and a standard HPLC-electrochemical method, requiring a larger volume of serum, has been determined. The two methods showed excellent agreement. Because of its low volume requirement, our HPLC-APCI assay can be substituted for the standard assay for the investigation of azithromycin pharmacokinetics in children.

Development and validation of a high-sensitivity assay for an antipsychotic agent, CP-88,059, with solid-phase extraction and narrow-bore high-performance liquid chromatography

An analytical method has been developed and validated for the quantitation of CP-88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C18 reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within +/- 10% and +/- 15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase.

Direct plasma injection for high-performance liquid chromatographic–mass spectrometric quantitation of the anxiolytic agent CP-93 393

A direct plasma injection method has been developed for the rapid analysis of drugs in biological fluids. A new generation restricted access media column specifically designed to accommodate direct injection of plasma and other fluids is utilized for on-line HPLC-ESI-MS analysis. For rapid analysis the on-line extraction column is linked to a HPLC-ESI-MS system. Good results are obtained for the quantitation of CP-93 393 and deuterated internal standard over the range of 10-1000 ng/ml. The lower limit of detection for the assay was 58 pg injected on column. Accuracy and precision values are 9.0% or better over the entire range of the assay. In addition, more than 200 injections (100 microl) were performed per column with unattended, automated analysis.

Robotics for the bioanalytical laboratory A flexible system for the analysis of drugs in biological fluids

Abstract The configuration of a robotic system for the complete automation of methods for trace level quantification of drugs in biological fluids is described. The system was designed to be applicable to numerous assays and to be rapidly switchable from one assay to the next. It employs a Zymate robot and various cost effective modules and components. The robot aliquots plasma and urine samples and performs dilution, mixing, precipitation, centrifugation, solvent extraction, evaporation, reconstitution and direct injection into a high-performance liquid chromatograph. An analytical report on each sample and a summary report on all samples are automatically produced for later incorporation into the data base. The effective reach of the robotic arm is limited to 1640 square inches of bench space. The bench space requirement is therefore an important criterion for the selection and design of robotic modules. A second important criterion is the failure rate. Because the present robot lacks the critical senses of touch and vision, some seemingly simple manual steps are difficult to automate. Effective substitutes for such steps have been devised. Some modifications of the manual method were required for effective automation. The robotic system was applied to several assays and the quality of the data generated was similar to or better than that of manual methods. The return on investment in robotic implementation is favorable.

Narrow-bore high-performance liquid chromatography in combination with ionspray tandem mass spectrometry for the determination of the substance P receptor antagonist ezlopitant and its two active metabolites in plasma

A simple, but highly sensitive and specific, assay was developed for the quantitative determination of ezlopitant and its two active metabolites in human plasma using narrow-bore reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ES-MS-MS). Ezlopitant, its two pharmacologically active metabolites, an alkene analogue (CJ-12 458) and a benzyl alcohol analogue (CJ-12 764), and their corresponding trideuterated internal standards (I.S.), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (60:40, v/v). Column effluent was monitored by pneumatically assisted electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ions was used to quantify ezlopitant and its two active metabolites. The assay exhibited a linear dynamic range of 0.1-100 ng/ml. Average absolute recoveries from plasma were approximately 71, 80 and 99% for ezlopitant and its two active metabolites CJ-12 485 and CJ-12 764, respectively. The precision (RSD %) and accuracy (Deviation %) values for the method were within +/- 12% and +/- 15%, respectively, for all analytes. Sample analysis times were less than 5 min from one injection to the next. The assay proved to be suitable for pharmacokinetics studies.

Development of a high-performance liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric assay for β-tigogenin cellobioside in human serum

A specific high-performance liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometric assay for the quantitative determination of beta-tigogenin cellobioside in human serum is described. Serum cleanup and acetylation of the analyte were required to achieve the desired lower limit of quantification, 10 ng/ml. The precision of the assay was better than 13% over a serum concentration range of 10-500 ng/ml.