Development and Validation of a Multiclass, Multiresidue Method for Veterinary Drug Analysis in Infant Formula and Related Ingredients Using UHPLC-MS/MS.

A multiclass, multiresidue method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed and validated for the analysis of around 150 veterinary drugs in infant formula and related dairy ingredients. The included analytes belong to the following veterinary drug classes: anthelmintics, antibiotics (aminoglycoside, amphenicols, ß-lactams-penicillins and cephalosporins, lincosamides, macrolides, quinolones, sulfonamides, tetracyclines, and others), antimicrobial growth promoters, antiprotozoals, ß-agonists, coccidiostats, dyes, pesticides, and tranquilizers. The sample preparation procedure involves dispersing the sample in 0.05 M EDTA solution in water, followed by extraction with 0.1% formic acid in acetonitrile, drying down an aliquot of the extract, and reconstituting it in a water-acetonitrile mixture. The analyte detection, identification, and quantitation are performed by UHPLC-MS/MS using positive electrospray ionization mode. The method was validated in infant formula powder, whole milk powder, and whey protein isolate, typically achieving limits of quantitation (meeting acceptable recovery and precision validation criteria) at 1-10 ng/g.

High-throughput bioanalysis of bile acids and their conjugates using UHPLC coupled to HRMS.

BACKGROUND: Quantitative assessment of bile acids in biological matrixes is of growing interest, primarily due to hepatic toxicity resulting from drug interactions with the bile salt export pump. Nevertheless, many bile acids demonstrate poor fragmentation in MS, making conventional MS/MS not a good match for their selective quantitation in biological matrices. RESULTS: The current study was designed to evaluate the feasibility of simultaneous quantitation of 19 bile acids using HRMS coupled to UHPLC separation with minimal instrument optimization. An effective chromatography was developed using an Agilent Zorbax(®) Eclipse XDB-C18 column (1.8 µm, 50 x 2.1 mm internal diameter), achieving separation of 19 compounds in 10 min. Excellent assay reproducibility was demonstrated, with two sets of standard curves, run 42 days apart. CONCLUSIONS: The results show that LC-HRMS is a viable platform for high throughput bioanalysis of bile acids especially in a drug-discovery setting.

Perforated dried blood spots: a novel format for accurate microsampling.

Dried blood spots (DBS) in their current format encounter challenges in bioanalysis using fixed areas, including but not limited to, waste of DBS samples (only a fraction is used for analysis), the need for sample punching leading to concerns of sample carryover, uncertainty for accurate recovery assessments and hematocrit (HCT) effects. Here we describe a novel concept, namely perforated dried blood spots (PDBS), for accurate microsampling that addresses previous challenges. PDBS discs were prepared from regular filter paper, with a diameter of 6.35 mm and a thickness of 0.83 mm. An accurate amount of blood sample (5-10 µl), was deposited, dried and stored on the PDBS discs. Upon sample analysis, PDBS samples are simply pushed by single-use pipette tips into 96-well plates. The proof-of-concept study was carried out on a PDBS LC-MS/MS assay development and validation under GLP criteria for the quantitation of lansoprazole in human whole-blood (K(3)EDTA). Particularly, the effect of HCT on the accuracy of quantitation was found to be related to recovery from PDBS samples. In all, PDBS was proved to be a viable alternative to conventional DBS, offering additional advantages of complete sample utilization, no requirement for punching, ease of recovery assessments, and elimination of sampling influence due to HCT levels.

LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots.

BACKGROUND: Dried blood spot (DBS) technology offers distinctive preclinical and clinical advantages primarily ascribed to microscale sampling (e.g., 40-80 µl per time point), and the nature of solid-state samples in filter papers. Logistic benefits in sample collection, storage and shipping also result. However, the effective DBS samples available for bioanalysis are finite, that is, in the order of approximately 1 µl equivalent of plasma (3-mm punch) from a DBS of approximately 15-20 µl whole blood samples. This represents 20- to 100-times fewer samples for bioanalysis compared with a typical plasma assay. It is critical to increase LC-MS/MS sensitivity to accommodate DBS bioanalysis. RESULTS: We developed a 2D strong cation exchange reversed-phase LC-MS/MS (2D-SCX/RPLC-MS/MS) for online enrichment, separation and detection of basic polar compounds, using clonidine hydrochloride as a model compound. Positively charged clonidine was retained and enriched in the first dimensional SCX column even in large volumes, eluted to a second dimensional RP column with ammonium acetate, de-salted with highly aqueous solvent and separated in an analytical RP column. Injection of 100 µl clonidine extract exhibited essentially the same peak shape as that from 1 µl and the response of clonidine increased quantitatively in the range of 1-100 µl. CONCLUSION: The method was successfully employed to analyze clonidine DBS samples from an in-house toxicology study, where clonidine hydrochloride was administered to cynomolgus monkeys to produce hypotensive effects. Of 55 DBS samples collected post-dose, a total of 52 samples were within the curve range of 0.1-50 ng/ml, where valid clonidine PK profiles were obtained. The PK parameters agreed well with the onset of hemodynamic changes measured with implanted miniature telemetry blood pressure transmitters. In comparison, only 21 samples were within the curve range of 2 to 1000 ng/ml from a HILIC-MS/MS method, which limited useful injection volume to 5 µl.

Liquid chromatography/tandem mass spectrometry sensitivity enhancement via online sample dilution and trapping: applications in microdosing and dried blood spot (DBS) bioanalysis.

A simple online sample dilution, enrichment, and cleanup technique was developed for sensitive microdosing and dried blood spot (DBS) liquid chromatography/tandem mass spectrometric (LC/MS/MS) bioanalysis. Samples are diluted online with water and enriched in a trap column which is subsequently switched inline with the analytical column. Excellent lansoprazole (in acetonitrile) peak shape is maintained even with an 80-microL injection. In comparison, similar chromatographic peaks were observed only when a small volume of the same solution, i.e., 1 microL, was injected on a regular high-performance liquid chromatography (HPLC) system, where an injection of 5 microL resulted in severe peak fronting. A substantial enhancement in sensitivity is realized in the trapping mode using large injection volumes. The trap column is washed at the beginning and at the end of each injection with aqueous and organic solvent respectively to remove matrix components. This ultimately leads to reduction of matrix effects and mass spectrometer noise, thus facilitating the utilization of protein precipitation as the sample preparation for plasma samples. A lower limit of quantitation (LLOQ) of 0.5 pg/mL was demonstrated for lansoprazole in human plasma with a signal-to-noise (S/N) ratio of 13 using a 100 microL injection. Excellent intra-day precision and accuracy were established for lansoprazole in human plasma with good linearity (R(2) > 0.999) from 0.5 to 500 pg/mL. This level of LLOQ makes LC/MS/MS a practical alternative for microdosing bioanalysis, where the dose is typically 100 times lower than the therapeutic dose. The same technique was applied to quantitate lansoprazole in human whole blood employing DBS technology. With a single 3-mm punch, i.e. approximately 2 microL of whole blood or approximately 1 microL plasma, a LLOQ of 0.1 ng/mL showed sufficient S/N ratio (40) for lansoprazole when 75 microL of extract was injected. In all, the online sample dilution, cleanup, and enrichment technique demonstrated the practical utility of LC/MS/MS in microdosing and DBS bioanalysis.