William Kemnitzer (Presenter)
DPX Labs
Authorship: Howard Horng (1) and William Kemnitzer (2)
(1) University of California, San Francisco (UCSF)/San Francisco General Hospital (SFGH), San Francisco, CA; (2) DPX Labs, Columbia, SC
| Short Abstract A novel method for the protein precipitation of serum was investigated using WAX dispersive pipette extraction (DPX) tips and analyzed by LC-MS-MS. Significantly, protein precipitation was performed within the DPX WAX tip to remove matrix interferences in less than 2 minutes. Viability was determined by testing the method on a comprehensive panel and comparing the results to standard protein precipitation preparations. Comparison between the two methods revealed that highly lipophilic compounds (LogP > 4) can be predicted to achieve a superior sample cleanup using the DPX WAX tips. |
Long Abstract
Introduction
Sample preparation is required to remove proteins from serum samples prior to LC-MS-MS analysis. The current method of choice involves “crashing” the proteins from serum. This is accomplished by typically adding acetonitrile or methanol to the sample, followed by vortex mixing and then centrifugation. The resulting supernatant is then transferred for downstream analysis. Although the protein precipitation method generally provides solid results, it is not an ideal solution for automated platforms.
Dispersive pipette extraction (DPX) tips are a solid phase extraction device designed for liquid handlers that incorporates a loosely contained sorbent in a pipette tip between a frit at the narrow bottom end of the tip and a barrier at the wider top end of the tip. Recently, it was reported that 25-OH vitamin D2 and D3 could be rapidly quantitated from serum using the DPX WAX tips without the need for centrifugation (1). Protein precipitation was accomplished within the DPX WAX tip providing a 60 second cleanup method. By using loose sorbent, matrix interferences were removed and trapped by the WAX resin without clogging the frit.
To further explore the application of DPX WAX tips in clinical toxicology, we have tested the 25-OH vitamin D2 and D3 method on a comprehensive panel of analytes. In addition, we have performed comparison studies between the DPX WAX tip and standard protein precipitation preparations. Preliminary results suggest that using the DPX WAX tip for protein removal can be an alternative to protein precipitation methods.
Methods
DPX WAX tip preparation involved aliquoting spiked serum sample (100 µl) into a culture tube and fentanyl-d5 (internal standard)/acetonitrile solution (300 µl) into a separate culture tube. Serum sample were aspirated into the DPX WAX tips followed by the aspiration of air into the bottom of the WAX tip. The fentanyl-d5/acetonitrile solution was then aspirated into the same WAX tip. The combined solution was then dispensed, re-aspirated and re-dispensed an additional two more times. The remaining solution was then collected and evaporated to dryness. Protein precipitation sample preparation involved aliquoting spiked serum sample (100 µl) into microcentrifuge tubes, followed by quenching with the fentanyl-d5/acetonitrile solution (300 µl), vortexing (1 min), and centrifuging (10,000 rpm) for 10 mins. The supernatant (800 µl) was transferred into a culture tube and evaporated down to dryness. The samples for both preparations was then reconstituted with mobile phase (100 µl) and injected onto the liquid chromatography-high resolution mass spectrometry (LC-HRMS) for analysis. Targeted analysis was carried out using a Shimadzu LC-20AD HPLC coupled to a AB Sciex Triple TOF 5600 high resolution mass spectrometry (HRMS) utilizing ESI positive ionization. HRMS analysis was performed on a reverse phase column (Phenomenex Kinetex, 2.6 µm, 50 x 3.0 mm) and a gradient system of 5.0 mM ammonium formate/0.050% formic acid in water (mobile phase A) and 50% acetonitrile/50% methanol (mobile phase B) over 14 minutes at a flow rate of 0.4 ml/min. Data was collected in full mass scan mode (MH+ m/z 50 to 700) with IDA-triggered acquisition of product ion scan. The data was analyzed using AB Sciex’s PeakView Software. Criteria for positive identification of a drug included chromatographic retention time (≤ 5% error), accurate mass (≤ 10 ppm mass error), isotope pattern (≤ 20% isotope ratio difference), and spectral library match (purity score ≥ 70).
Results
The analysis of the sample preparation recoveries of 39 drugs (DPX versus protein precipitation) were classified into the following three categories: class 1 (DPX tip recovery is > 20% than that of protein precipitation), class 2 (DPX tip recovery is equal (±20%) to that of protein precipitation), and class 3 (DPX tips recovery is < 20% than that of protein precipitation). Class 1 included the drugs clomipramine, desipramine, cyclobenzaprine, and haloperidol. Class 2 comprised of the drugs doxepine, desalkylflurazepam, quetiapine, gabapentin, hydrocodone, cimetidine, risperidone, zolpidem, amitriptyline, ziprasidone, citalopram, dextromethorphan, clonazepam, flunitrazepam, brompheniramine, and methylenedioxyphenethylamine (MDDMA). Class 3 included the drugs atropine, dihydrocodeine, oxycodone, codeine, phentermine, diazepam, loxapine, methadone, methylphenidate, misanserine, venlafaxine, clobazam, clozapine, naloxone, phenytoin, benzyoylecgonine, levamisole, morphine, and mefenamic acid. Analysis of the physiochemical properties of each class reveals that class 1 drugs are all highly lipophilic (LogP > 4). While the lipophilicity of class 2 and class 3 drugs remain similar (LogP ~ 2-3). The exception to this rule are doxepin (class 2; LogP 4.29), amitriptyline hydrochloride (class 2; LogP 4.92), and mefenamic acid (class 3; LogP 5.12).
Conclusion
DPX WAX tips provide a fast, accurate, and simple extraction technique for analyzing a number of toxicological compounds in serum through the removal of matrix interferences associated from acetonitrile quenched samples. Highly lipophilic compounds (LogP > 4) are predicted to achieve a superior sample cleanup using the DPX WAX tips. The acidic compounds, mefenamic acid and amitriptyline hydrochloride, are shown to be the exception to this rule. However, compounds with intermediate lipophilicity may achieve recoveries similar to that of protein precipitation preparations.
References & Acknowledgements:
(1) Woodward, G.; Brewer, W.; Roch, M.; Webster, R., “Rapid Quantitative Analysis of 25-OH Vitamin D2 & D3 in Patient Serum using a Novel Weak Anion Exchange Disposable Tip Extraction (DPX) and LC-MS/MS Detection,” MSACL 2015, San Diego, CA.
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