Quantification of midazolam, morphine and metabolites in plasma using 96‐well solid‐phase extraction and ultra‐performance liquid chromatography–tandem mass spectrometry

Abstract

Currently, pharmacokinetic-pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1-hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine-3-glucuronide and morphine-6-glucuronide in 100 microL of plasma. Cleanup consisted of 96 wells micro-solid phase extraction, before reversed-phase chromatographic separation (ultra-performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid-phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra- and interrun accuracy and precision were within 85-115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96-well micro-SPE and UPLC-MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 microL of plasma.

High performance liquid chromatographic assay for the simultaneous determination of midazolam and ketoconazole in plasma

“…Precision was determined using percentage coefficient of variation (CV%) and bias was assessed using mean intra-or interday percentage error of the mean. The stability of MDZ and KTZ in working solutions and plasma has been previously described extensively [19][20][21].…”

Microextraction Techniques in Illicit Drug Testing: Present and Future

“…DPX was also described in the 96-well plates format for high-throughput cleanup [14]. An additional improvement of MEPS for OF testing has been described in a recent article and consists of the introduction of a catheter to aspirate saliva directly from the mouth into the MEPS syringe [15].…”

Individualized dosing regimens in children based on population PKPD modelling: Are we ready for it?

“…Meanwhile modern technologies for blood sampling and laboratory analyses have been developed which facilitate studies in paediatric populations. LC-MS/MS, for example, is a highly sensitive analytical technique enabling the use of very small volume samples for drug concentration analyses (Ahsman et al, 2010(Ahsman et al, , 2009). In addition to these novel technologies, the application of advanced data-analysis techniques, the so-called 'population approach' has opened new avenues for drug studies in children.…”