A review of current trends and advances in modern bio-analytical methods: chromatography and sample preparation.

Tutorial review on validation of liquid chromatography-mass spectrometry methods: part II.

Lactoferrin (LF) is a multifunctional protein occurring in many biological secretions including milk. It possesses iron binding/transferring, antibacterial, antiviral, antifungal, anti-inflammatory and anti-carcinogenic properties. These functional properties intimately depend on the structural integrity of LF especially its higher order conformation. LF is primarily extracted from bovine milk and it is subsequently added into many commercial products such as nutritional supplements, infant formula, cosmetics and toothpaste. LF is sensitive to denaturation induced by temperature and other physicochemical stresses. Hence, the extraction, powder formation processes of LF and processing parameters of LF-containing products have to be optimized to minimise its undesired denaturation. This review documents the advances made on structure-function relationships and discusses the effectiveness of methods used to preserve the structure of LF during thermal processing. Oral delivery, as the most convenient way for administering LF, is also discussed focusing on digestion of LF in oral, gastric and intestinal stages. The effectiveness of methods used to deliver LF to intestinal digestion stage in structurally intact form is also compared. Altogether, this work comprehensively reviews the fate of LF during thermal processing and digestion, and suggests suitable means to preserve its structural integrity and functional properties. Scope of review The manuscript aims at providing a comprehensive review of the latest publications on four aspects of LF: structural features, functional properties, nature and extent of denaturation and gastrointestinal digestion. It also analyses how these publications benefit food and pharmaceutical industries.

Lactoferrin: Structure, function, denaturation and digestion

In recent years, different approaches have been taken to improve chromatographic performance in terms of analysis time and/or resolution. The use of columns packed with sub-2μm particles in ultra-high-pressure liquid chromatography (UHPLC) has become a technique of choice in many laboratories. Furthermore, for the analysis of complex matrices (e.g., biological fluids, plant extracts, and food and environmental samples), coupling UHPLC with mass spectrometry (MS) or tandem MS (MS 2 ) provides a powerful analytical tool. This review describes major advances in the field of UHPLC-MS and UHPLC-MS 2 . We strongly emphasize the possibility of speeding up bioanalysis, drug metabolism, and multi-residue screening assays, while maintaining qualitative and quantitative performance equivalent to HPLC-MS and HPLC-MS 2 . We also report the possibility of gaining additional information in metabolomics, using high-resolution UHPLC with a time-of-flight analyzer. The studies summarized are discussed in this review in terms of throughput increases and resolution enhancements afforded by UHPLC. In addition, we highlight the impact of UHPLC conditions on MS detection capabilities (e.g., acquisition rate, limits of detection and matrix effects).

Coupling ultra-high-pressure liquid chromatography with mass spectrometry

The AAPS Workshop 2008 on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples was the defining moment in establishing incurred sample reanalysis (ISR) as a mandatory exercise in demonstrating assay reproducibility using incurred (study) samples. The importance of ISR can be envisaged from its role in clinical as well as non-clinical studies. Incurred samples can differ significantly in their composition when compared with the calibration standards and quality control samples that are used to validate the developed method. The present article attempts to summarize five troubleshooting cases encountered in the analyses of incurred samples for bioanalytical methods developed in our laboratory for mesalamine, hydrochlorothiazide, clopidogrel, sildenafil and rabeprazole. The issues identified were related to: sample inhomogeneity, sample processing error, impact of buffer pH during sample preparation, instability of metabolite and change in laboratory environment. The steps taken to trace and correct these incidents are discussed with adequate data. These examples will further broaden the scope and emphasize the significance of ISR. We believe this investigation will help to develop more reliable and efficient bioanalytical methods.

Incurred sample reanalysis (ISR): a decisive tool in bioanalytical research

A simple, precise, and rapid LC/MS/MS method was developed and validated for the quantification of emtricitabine in human plasma using lamivudine as the internal standard (IS). The method involves liquid-liquid extraction of emtricitabine and the IS in diethyl ether-ethyl acetate (50 + 50, v/v) from 0.1 mL human plasma. A Kromasil C18 (150 x 4.6 mm, 5 microm particle size) analytical column was used for the chromatographic separation under isocratic conditions. The parent --> product ion transitions for emtricitabine (m/z 248.0 --> 130.0) and the IS (m/z 230.1 --> 112.0) were monitored on a triple-quadrupole mass spectrometer, operated in the multiple reaction monitoring positive ion mode. The method was validated over the concentration range of 29.2-3110 ng/mL for emtricitabine, with a total chromatographic run time of 1.5 min. Acceptable precision and accuracy were obtained for the concentrations used to prepare the standard curves. The applicability of the method was demonstrated by a pharmacokinetic study conducted with 43 healthy volunteers who were administered a capsule formulation containing 200 mg emtricitabine.

Microdetermination of emtricitabine in human plasma by liquid chromatography/tandem mass spectrometry.

Objective: The work presents a sensitive, selective and rapid determination of lapatinib, a potent anticancer drug in human plasma by liquid chromatography-tandem mass spectrometry. Methods: Liquid-liquid extraction of lapatinib and lapatinib-d4, added as an internal standard (IS) was carried out from 100 µl plasma sample. Chromatographic analysis was performed on ACE C18 (100 mm × 4.6 mm, 5 µm) column using 10 mmol ammonium formate buffer (pH 3.5) and acetonitrile (10:90, v/v) as the mobile phase. The precursor ion → product ion transitions for lapatinib (m/z 581.1 → 365.2) and IS (m/z 585.1 → 365.0) were monitored on a triple quadrupole mass spectrometer in the positive electrospray ionization mode. The method was validated in accordance with the US FDA guidelines. Results: A linear concentration range was established from 2.50-2500 ng/ml for lapatinib. The intra-batch and inter-batch precision were ≤ 4.81 %. The recovery of lapatinib and IS from plasma samples ranged from 88.7 to 95.8 % and 85.9 to 96.5 % respectively. The accuracy and precision (% CV) for the stability of lapatinib under different storage conditions showed a variation from 95.2 to 102.2 % and 1.19 to 4.35 % respectively at low and high QC levels. Under optimized chromatographic conditions, the retention time for lapatinib was 1.406 min with a total run time of 2.5 min for each sample. Conclusion: The validation results demonstrate that the method is simple, accurate, precise and reproducible. The developed method can be readily used for pharmacokinetics/bioequivalence studies in patients as well as healthy subjects.

Puran Singhal

Papers

Microdetermination of emtricitabine in human plasma by liquid chromatography/tandem mass spectrometry.

SENSITIVE AND RAPID ESTIMATION OF LAPATINIB, AN ANTICANCER DRUG IN SPIKED HUMAN PLASMA BY LC-MS/MS Original Article