Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

Determination of active vitamin B12 (cobalamin) in dietary supplements and ingredients by reversed-phase liquid chromatography: Single-laboratory validation.

Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w-85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses.

Improved Chiral Separation of Methamphetamine Enantiomers Using CSP-LC-MS-MS.

To determine the true enantiomeric composition of methamphetamine urine drug testing results, chiral separation of dextro (D) and levo (L) enantiomers is necessary. While enantiomeric separation of methamphetamine has traditionally been accomplished using gas chromatography-mass spectrometry (GC-MS), chiral separation of D- and L-methamphetamine by chiral stationary phase (CSP) liquid chromatography-mass spectrometry/mass spectrometry (LC-MS-MS) has proved more reliable. Chirally selective detection of methamphetamine by GC-MS is often performed using L-N-trifluoroacetyl-prolyl chloride (TPC). L-TPC, a chiral compound, is known to have impurities that can affect the chiral composition percentages of the methamphetamine sample, potentially leading to inaccurate patient results. The comparative analysis of the samples run by GC and LC methods showed preferential bias of the GC method for producing error rates, consistent with previous research, of 8-19%. The CSP-LC-MS-MS method produces percent deviation errors of <2%. Additionally, the GC method failed to produce results that were 100% D- or L-isomer even for enantiomerically pure standards. A higher rate of D- and L-methamphetamine isomer racemization is seen in samples when analyzed by GC-MS using L-TPC-derivatizing agent. This racemization is not seen when these samples are tested with CSP-LC-MS-MS. Thus, a more accurate method of enantiomeric analysis is provided by CSP-LC-MS-MS.

Rational method development strategies on a fluorinated liquid chromatography stationary phase: mobile phase ion concentration and temperature effects on the separation of ephedrine alkaloids.

Fluorinated, silica-based stationary phases are becoming increasingly popular alternatives to traditional alkyl phases owing to their differential selectivity and retention for a variety of analyte classes. In this report, the ion-exchange mechanisms characteristic of a fluorinated phase are exploited to rapidly develop separation conditions for ephedrine alkaloids and synephrine using a mobile phase compatible with mass spectrometry. A linear relationship of basic analyte retention with the reciprocal of ammonium acetate concentration is first established. This linear relationship can then be used to optimize retention and selectivity in just two experiments. The relationship of retention with temperature is also explored. Greater retention with increasing temperature is demonstrated on the fluorinated phase at high percentages of organic modifier, which is in contrast to behavior observed in typical reversed-phase separations. The unexpected observation is explicated based on the reduction in solvent solvating power with increasing temperature. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger interactions between analyte and stationary phase. Both mobile phase ion concentration and temperature are shown to be powerful tools for the manipulation of analyte retention and selectivity.