Liquid-phase microextraction in 96-well plates - calibration and accurate quantification of pharmaceuticals in human plasma samples.

The use of miniaturized systems with the possibility for automation has become increasingly popular in the field of bioanalysis. As a new approach to liquid phase microextraction in the 96-well format, parallel artificial liquid membrane extraction (PALME) was introduced in 2013. In the present work, the reliability of the quantitative data obtained with PALME was thoroughly evaluated. Amitriptyline, nortriptyline, quetiapine, venlafaxine, o-desmethylvenlafaxine, and fluoxetine were selected as model analytes. The analytes were extracted under non-exhaustive conditions from human plasma samples and the extracts were analyzed directly by LC-MS/MS. Accuracy was within ±15% and precision was <15% when the QC samples were prepared in both pooled plasma and in plasma from multiple sources. Accuracy and precision was superior when stable isotopically labelled (SIL) internal standards were used, as compared to structural analogue internal standards in the plasma samples from multiple sources. SIL internal standards are therefore recommended as the first choice. Assessment of accuracy and precision was also carried out with four different operators performing the extraction procedure, providing accuracy within ±15% and precision <15%. The extraction recoveries were in the range from 48 to 85 %, and non-exhaustive extraction of the analytes did not affect the accuracy and precision of the method. With the method described, up to 96 samples can be extracted with a total extraction time of 60 min and with a total consumption of organic solvent less than 0.4 mL for the whole wellplate. PALME is therefore a new approach to high-throughput sample preparation, providing accurate quantification, along with simple workflow, low consumption of organic solvent, and extensive sample clean-up.

Dried blood spots and parallel artificial liquid membrane extraction-A simple combination of microsampling and microextraction.

In this paper, parallel artificial liquid membrane extraction (PALME) was used for the first time to clean-up dried blood spots (DBS) prior to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Fundamental studies exploring amongst others desorption from the DBS in alkaline or acidic aqueous conditions, total extraction time and absolute recoveries were executed. Desorption and PALME were performed using a set of two 96-well plates, one of them housing the sample and the other comprising the supported liquid membrane (SLM) and the acceptor solution. In one procedure, amitriptyline and quetiapine (basic model analytes) were desorbed from the DBS using 250 µL of 10 mM sodium hydroxide solution (aqueous), and subsequently extracted through the SLM consisting of 4 µL of 1% trioctylamine in dodecyl acetate, and further into an acceptor solution consisting of 50 µL of 20 mM formic acid. In a second procedure, ketoprofen, fenoprofen, flurbiprofen, and ibuprofen (acidic model analytes) were desorbed from the DBS into 20 mM formic acid, extracted through an SLM with dihexyl ether, and further into an acceptor solution of 25 mM ammonia. Within 60 min of PALME, both basic and acidic model analytes were effectively desorbed from the DBS and extracted into the acceptor solution, which was injected directly into the analytical instrument. Recoveries between 63 and 85% for the six model analytes were obtained. PALME provided excellent clean-up from the DBS samples, and acceptor solutions were free from phospholipids. Linearity was obtained with r2 > 0.99 for five of the six analytes. Accuracy, precision and UHPLC-MS/MS matrix effects were in accordance with the European Medicines Agency (EMA) guideline. Based on these experiments, PALME shows great potential for future processing of DBS in a short and simple way, and with the presented setup, up to 96 DBS can be processed within a total extraction time of 60 min.

Parallel artificial liquid membrane extraction of psychoactive analytes: a novel approach in therapeutic drug monitoring.

AIM: Liquid-liquid extraction is widely used in therapeutic drug monitoring of antipsychotics, but difficulties in automation of the technique can result in long operational time. In this paper, parallel artificial liquid membrane extraction was used for extraction of serotonin- and serotonin-norepinephrine reuptake inhibitors from human plasma, and an approach to automate the technique was investigated. RESULTS: Eight model analytes were extracted from 125 µl human plasma with recoveries in the range 72-111% (relative standard deviation [RSD] ≤12.8%). A semiautomated pipettor was successfully utilized in the procedure, reducing the manual handling time. Real patient samples were analyzed with satisfying accuracy. CONCLUSION: A semiautomated extraction of serotonin-and serotonin-norepinephrine reuptake inhibitors by parallel artificial liquid membrane extraction extraction was successfully performed.

One-step extraction of polar drugs from plasma by parallel artificial liquid membrane extraction.

The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid-liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5µL per sample. With a sample volume of 250µL and acceptor solution volume of 50µL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50-89% from human plasma after 45min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs.

Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125µL), through 4µL organic solvent used as supported liquid membranes (SLMs), and into 50µL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines.