tacrolimus and acetonitrile

In preparative chromatography, often the solubility of the sample in the mobile phase is limited, making the mobile phase unsuitable as a solvent for preparation of load. Generally, solvents that have high solubility for the sample also have higher elution strengths than the mobile phase. Additionally, at high loading volumes, these strong sample solvents are known to adversely affect the band profiles leading to poor chromatographic performance. Here, we show that controlling the mobile phase strength during loading and post-load elution resulted in improved band profiles when the sample solvent was stronger than the mobile phase. Such an approach improves performance in preparative chromatography by allowing either higher sample loading or higher organic content in mobile phase (without loss of yield). Alternately, the approach can be used for improvement in performance by increase in yield or product purity.

Topics: Acetonitriles; Adsorption; Chromatography, High Pressure Liquid; Hydrophobic and Hydrophilic Interactions; Methanol; Reproducibility of Results; Sensitivity and Specificity; Silicon Dioxide; Solubility; Solvents; Spectrophotometry, Ultraviolet; Stereoisomerism; Tacrolimus; Water

Topics: Acetonitriles; Chromatography, Liquid; Humans; Immunosuppressive Agents; Mass Spectrometry; Methanol; Quality Control; Reference Standards; Solvents; Tacrolimus

In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 microgram/L. The interassay variability was 14.5% for 5 micrograms/L and 15.8% for 25 micrograms/L. The intrassay variability was 11.2% for 5 micrograms/L and 4% for 25 micrograms/L. The recovery for tacrolimus in blood was 90.4% for 1 microgram/L, 78.9% for 10 micrograms/L, and 81.3% for 25 micrograms/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, di-demethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.

Topics: Acetonitriles; Calibration; Chromatography, High Pressure Liquid; Humans; Immunoenzyme Techniques; Immunosuppressive Agents; Kidney Transplantation; Liver Transplantation; Mass Spectrometry; Reference Standards; Reproducibility of Results; Tacrolimus; Water