Francis L.S. Tse

Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules.

The collection of whole blood samples on paper, known as dried blood spot (DBS), dates back to the early 1960s in newborn screening for inherited metabolic disorders. DBS offers a number of advantages over conventional blood collection. As a less invasive sampling method, DBS offers simpler sample collection and storage and easier transfer, with reduced infection risk of various pathogens, and requires a smaller blood volume. To date, DBS-LC-MS/MS has emerged as an important method for quantitative analysis of small molecules. Despite the increasing popularity of DBS-LC-MS/MS, the method has its limitations in assay sensitivity due to the small sample size. Sample quality is often a concern. Systematic assessment on the potential impact of various blood sample properties on accurate quantification of analyte of interest is necessary. Whereas most analytes may be stable on DBS, unstable compounds present another challenge for DBS as enzyme inhibitors cannot be conveniently mixed during sample collection. Improvements on the chemistry of DBS card are desirable. In addition to capturing many representative DBS-LS-MS/MS applications, this review highlights some important aspects of developing and validating a rugged DBS-LC-MS/MS method for quantitative analysis of small molecules along with DBS sample collection, processing and storage.

High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main metabolite (CGP 74588) in human plasma using liquid chromatography-tandem mass spectrometry.

The signal transduction inhibitor STI571 (formerly known as CGP 57148B) or Gleevec received fast track approval by the US Food and Drug Administration (FDA) for treatment of chronic myeloid leukemia (CML). STI571 is a revolutionary and promising new oral therapy for CML, which functions at the molecular level with high specificity. The dramatic improvement in efficacy compared to existing treatments prompted an equally profound increase in the pace of development of Gleevec. The duration from first dose in man to completion of the New Drug Application (NDA) filing was approximately 2.6 years. In order to support all pharmacokinetics studies with sufficient speed to meet various target dates, a semi-automated procedure using protein precipitation was developed and validated. A Tomtec Quadra 96 (Model 320) and a protein precipitation step in a 96-well plate format were utilized. A Sciex API 3000 triple quadrupole mass spectrometer with an atmospheric pressure chemical ionization interface operated in positive ion mode was used for detection. The method proved to be rugged and allowed the simultaneous quantification of STI571 and its main metabolite (CGP 74588) in human plasma. Herein, assay development, validation, and representative concentration-time profiles obtained from clinical studies are presented.

Quantification of the anti-leukemia drug STI571 (Gleevec) and its metabolite (CGP 74588) in monkey plasma using a semi-automated solid phase extraction procedure and liquid chromatography-tandem mass spectrometry.

Signal Transduction Inhibitor 571 (STI571, formerly known as CGP 57148B) or Gleevec received fast track approval by the US Food and Drug Administration (FDA) for treatment of chronic myeloid leukemia (CML). STI571 (Gleevec) is a revolutionary and promising new oral therapy for CML, which functions at the molecular level with high specificity. The dramatic improvement in efficacy compared with existing treatments prompted an equally profound increase in the pace of development of Gleevec. The duration from first dose in man to completion of the New Drug Application (NDA) filing was less than 3 years. In addition, recently, FDA approved Gleevec for the treatment of gastrointestinal stromal tumor (GIST). In order to support all toxicokinetic (TK) studies with sufficient speed to meet various target dates, a semi-automated procedure using solid phase extraction (SPE) was developed and validated. A Packard Multi-Probe I and a SPE step in a 96-well plate format were utilized. A 3M Empore octyl (C(8))-standard density 96-well plate was used for plasma sample extraction. A Sciex API 3000 triple quadrupole mass spectrometer with an atmospheric pressure chemical ionization (APCI) interface operated in positive ion mode was used for detection. Lower limits of quantification of 1.00 and 2.00 ng/ml were attained for STI571 and its metabolite, CGP 74588, respectively. The method proved to be rugged and allowed the simultaneous quantification of STI571 and CGP 74588 in monkey plasma. Herein, assay development, validation, and representative concentration-time profiles obtained from TK studies are presented.

Liquid-liquid extraction using 96-well plate format in conjunction with liquid chromatography/tandem mass spectrometry for quantitative determination of methylphenidate (Ritalin) in human plasma.

Methylphenidate (MPH; Ritalin: methyl-alpha-phenyl-2-piperidinacetate hydrochloride) is utilized for the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy. Recently, we described a rapid enantioselective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of the enantiomers of MPH (Rapid Commun. Mass Spectrom. 1999; 13: 2054). A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. The present paper describes a high-throughput sample preparation procedure in conjunction with racemic LC/MS/MS analysis for MPH with a LLOQ of 50 pg/mL. A semi-automated robotics method using liquid-liquid extraction (LLE) in a 96-well plate format was developed and validated. The correlation coefficients were > or =0.998 for MPH indicating good fits of the regression models over the range of the calibration curve. The accuracy and precision of the semi-automated approach were comparable to those obtained using the manual sample preparation technique reported previously (vide supra). The current method can easily be adapted to the enantioselective LC/MS/MS assay of MPH. The assay was simple, fast, specific, and exhibited excellent ruggedness.

High-throughput chiral liquid chromatography/tandem mass spectrometry

Chiral liquid chromatography is a well-established area of bioanalytical chemistry and is often used during the processes of drug discovery and development. The development and use of a chiral drug require the understanding of the pharmacokinetic characteristics of each of the enantiomers, including potential differences in their absorption, distribution, metabolism, and excretion. Chromatographic techniques coupled to atmospheric pressure ionization-tandem mass spectrometry have shown potential as sensitive and robust tools in the quantitative and qualitative determination of enantiomers in biologic fluids and tissue extracts. However, development of a chiral liquid chromatography method requires time-consuming procedures that are devised empirically. Clearly, there is an incentive to design chromatographic approaches that are easy to use, compatible with mass spectrometry ionization interface conditions, exhibit relatively short run times without compromising sensitivity, and offer a broad analyte specificity. For these reasons, the present paper explores the feasibility of the bonded macrocyclic glycopeptide phases (teicoplanin and vancomycin) for analysis by chiral liquid chromatography/tandem mass spectrometry. Ritalinic acid, pindolol, fluoxetine, oxazepam, propranolol, terbutaline, metoprolol, and nicardipine were tested in this study. Furthermore, an example of a simultaneous chiral LC/MS/MS detection (chromatographic run time approximately 10 min) of four pharmaceutical products resulting in baseline resolutions of all four pairs of enantiomers is presented. Methanol, an MS-compatible mobile phase, was utilized in all the experiments.

Quantitative analysis of terbinafine (Lamisil) in human and minipig plasma by liquid chromatography tandem mass spectrometry.

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of terbinafine in human and minipig plasma has been developed and validated. The method used positive-ion mode for monitoring terbinafine, and used a stable isotope labelled terbinafine as the internal standard. Subsequent to acetonitrile protein precipitation, the supernatant was directly (unfiltered) injected onto the LC column (retention time approximately 4.3 min) for analysis. Interday and intraday accuracy and precision were assessed from the relative recoveries (observed concentration in percent of the nominal value) of spiked samples analyzed on three different days. The lower limit of quantitation (LLOQ) was 0.0679 ng/mL in human and minipig using a plasma sample volume of 0.08 mL. The method was fast, specific, and exhibited ruggedness. Furthermore, the use of turbulent flow chromatography (TurboFlow LC/MS/MS) coupled to mass spectrometry for direct analysis of terbinafine in plasma is discussed. The technique allowed direct introduction of plasma with satisfactory chromatographic peak shape and increased throughput.

Brain uptake of dihydroergotamine after intravenous and nasal administration in the rat.

This study was conducted to determine the uptake of dihydroergotamine (DHE) into the brain after intravenous and intranasal administration in rats. Eight anesthetized rats received either an intravenous (i.v.) or two successive intranasal (i.n.) doses of tritium labeled dihydroergotamine (3H‐DHE) with 14C‐inulin as a non‐BBB (blood–brain barrier) permeable marker. Radioactivity concentrations in plasma were determined at designated times within 30 min postdose, and in blood and seven brain regions (olfactory bulb, frontal cortex, parietal cortex, occipital cortex, cerebellum, mid‐brain areas, and brain stem) at 30 min. The plasma‐to‐brain permeability*area product (PeA) following an i.v. dose was calculated based on the 30‐min brain tissue concentration and the area under the plasma concentration–time curve (AUC0–30 min, i.v.) assuming unidirectional transport from plasma to brain. Direct transport from nasal cavity to brain was assessed based on the amount of radioactivity in brain determined experimentally and predicted based on plasma AUC0–30 min, i.n. and PeA obtained from i.v. data. Following an i.v. dose, DHE distributed into the brain with a brain‐to‐plasma concentration ratio of ∼5% at 30 min postdose. The PeA value of DHE ranged from 8.6×10−4 to 37.5×10−4 mL min−1 g−1 in different brain regions. Following i.n. doses the experimentally determined concentration in olfactory bulb was approximately 51 times, and in other regions three to seven times, greater than predicted values based only on PeA and plasma AUC, suggesting a direct transport pathway from the nasal cavity to the brain. As a result, the brain tissue concentrations at 30 min were similar to (0.31–1.04 times) those following an i.v. dose except for the olfactory bulb, in which the concentration was approximately four times greater than that following an i.v. dose. In conclusion, 3H‐DHE penetrated the BBB following intravenous administration. Following i.n. doses, 3H‐DHE was able to enter the brain directly from the nasal cavity, with the olfactory bulb being a part of the direct passage from nasal cavity to brain. Copyright

Liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry enantiomeric separation of dl-threo-methylphenidate, (Ritalin) using a macrocyclic antibiotic as the chiral selector.

Vancomycin, a macrocyclic antibiotic, is an amphoteric glycopeptide produced by Streptomyces orientalis which has proven to be a viable chiral selector for high performance liquid chromatograph (HPLC) (D. W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill and J-R. Chen, Anal. Chem. (1994; 66: 1473). While it is related to other glycopeptide antibiotics, vancomycin has a number of unique structural features, including 18 stereogenic centers, five aromatic rings, and two side chains one of which is a carbohydrate dimer. Therefore, a vancomycin-based stationary phase appears to be multimodal in that it can be utilized in both normal-phase and reversed-phase liquid chromatography. Consequently, the enantiomeric separation may be operative via several mechanisms, including pi-pi complexation, dipole stacking, inclusion, hydrogen bonding, or combinations of these interactions. LC/MS/MS is a powerful tool for quantitative analysis when evaluated on the basis of speed, specificity, reliability and sensitivity. For these reasons, the present paper explored the feasibility of bonded macrocyclic glycopeptide phases for chiral LC/MS/MS quantitative analysis. Methylphenidate was used as a model compound. A rapid chiral bioanalytical method (<7.5 min) for the determination of the enantiomers of methylphenidate was developed. A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. This is to our knowledge the first example of enantioselective reversed-phase LC/MS/MS for methylphenidate. The chiral column was relatively cost effective and exhibited excellent performance with no separation deterioration observed after approximately 2500 injections.

High-throughput approaches to the quantitative analysis of ketoconazole, a potent inhibitor of cytochrome P450 3A4, in human plasma.

Ketoconazole, an imidazole-piperazine compound, is an orally active antimycotic agent. In addition, ketoconazole is a specific inhibitor of cytochrome P450 3A4. As about 60% of oxidized drugs are biotransformed by this isoform, the potential effect of a concomitant administration of ketoconazole on drug disposition may be of interest during drug development. The present paper describes three different approaches (methods A, B, and C) to attain high-throughput sample preparation and analysis in the quantification of ketoconazole in human plasma. Method A consisted of acetonitrile precipitation in a 96-well plate, transfer of the supernatant via a Tomtec Quadra 96 Model 320, and subsequent injection onto a 50 x 4.6 mm (i.d.) Develosil Combi-RP-5 column (packed with C30 bonded silica particles). Method B consisted of an identical sample preparation to method A with the exception that a Michrom Magic Bullet(trade mark) column, 2.0 --> 0.50 mm (i.d., tapered bore) x25 mm length, was used. Lastly, in method C, a turbulent-flow chromatography (TurboFlow LC/APCI-MS/MS) module was used for the direct analysis of ketoconazole in human plasma. A Sciex API 3000 was used in methods A and B, while a Micromass Quattro LC was employed in method C. Based on the values obtained for the calibrator (standard) and quality control samples, all three protocols yielded satisfactory accuracy, precision, and reduced manual sample preparation time.

A sensitive and high-throughput LC–MS/MS method for the quantification of pegylated-interferon-α2a in human serum using monolithic C18 solid phase extraction for enrichment

The analysis of pegylated-interferon-alpha(2a) in patient serum samples is of high interest for clinic research trials, as this therapeutic protein has become an important antiviral treatment. In this study, an LC-MS/MS method for the absolute quantification of pegylated-interferon-alpha(2a) in human serum was developed. The assay achieved a lower limit of quantification of 3.6 ng/mL (60 pM) with the use of a monolithic C(18) solid phase extraction to enrich the target protein. The linear range of the assay was defined up to 54 ng/mL to measure the typical clinical pegylated-interferon-alpha(2a) levels, and within this range, the precision and accuracy were found to be within +/-20%. The method was applied to a clinical study and found suitable for high-throughput analysis of pegylated-interferon-alpha(2a) in human serum. In addition, further investigations suggested the enrichment step may have general application to the sensitive analysis of other low molecular weight proteins.