65th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics
RESTEK TECHNICAL PRESENTATIONS
Forensics II, TP 226
The Advantages of Direct Analysis of Morphine and Its Metabolites with Related Compounds in Urine by LC-MS/MS
Justin Steimling (presenter), Frances Carroll, Susan Steinike
For more information, e-mail Justin Steimling.
Introduction: The analysis of total morphine is typically conducted by first subjecting urine samples to acid or enzymatic hydrolysis in order to cleave the glucuronide conjugate from the parent drug prior to analysis. With the glucuronide moiety removed, morphine is much less polar and is, therefore, more amenable to traditional reversed-phase chromatography. Both hydrolysis procedures cost the analyst time and introduce a large source of error into the analysis. In this presentation, we will examine the benefit of direct analysis of morphine and its metabolites with related compounds in urine following a simplified and robust “dilute-and-shoot” sample preparation method.
Methods: Calibration and quality control samples were prepared by fortifying human urine with morphine, morphine-3β-D-glucuronide (M3G), morphine-6β-D-glucuronide (M6G), morphine-N-oxide, 6-MAM, and hydrocodone. A simple “dilute-and-shoot” sample preparation procedure was compared to two sample preparation methods, each using a different source of β-glucuronidase for hydrolysis. The samples subjected to enzyme hydrolysis were spiked with 750 ng/mL of each compound in order to monitor conversion efficiency. The results of the samples subjected to hydrolysis were quantitated using a calibration curve prepared in an enzyme solution surrogate in order to evaluate the bias resulting from enzyme hydrolysis. LC-MS/MS analysis was performed on a Shimadzu Nexera UHPLC equipped with a SCIEX API 4500 MS/MS using a Restek Force Biphenyl analytical column.
Preliminary Data: Linearity was evaluated in the range of 25-2,500 ng/mL for all analytes. Using 1/x2 weighted linear regression for morphine-N-oxide, and 1/x weighted linear regression for the remaining analytes, all compounds showed good linearity with r2 values of 0.996 or greater with relative standard deviations <10%. The lowest signal-to-noise ratio observed at the LLOQ across all analytes was >49:1, indicating that this method could be modified to increase the dilution factor or decrease the LLOQ as necessary. Analysis of blank urine showed no observed matrix interferences. Chromatographic resolution was achieved between the isobars M3G and M6G, and retention factors greater than 1.5 were observed for all compounds.
After 16 hours of incubation at 60 °C, it was observed that >99% of M3G had been converted into morphine for both enzymes. Purified β-glucuronidase from abalone converted >99% of M6G into morphine while only 80% was converted by the less pure enzyme. For both enzymes, only 400 ng/mL of 6-MAM was detected, indicating a significant 6-MAM to morphine conversion. As a result of the 6-MAM to morphine conversion, the purified enzyme resulted in a high recovery of morphine, while the less pure enzyme was much closer to the nominal value due to the incomplete hydrolysis of M6G. Interestingly, both enzymes resulted in the detection of elevated levels of hydrocodone (940 ng/mL). In this case, analysis by hydrolysis produced inaccurate results for total morphine, 6-MAM, and hydrocodone.
Novel Aspect: The direct analysis of total morphine levels without the need for hydrolysis.
Clinical Analysis II, ThP 074
Direct Injection of Antiretroviral Drugs in Highly Organic Protein-Precipitated Human Plasma by LC-MS/MS
Frances Carroll (presenter), Justin Steimling, Susan Steinike
For more information, e-mail Frances Carroll.
Introduction: Protein precipitation (PPT) extraction is widely used to prepare biological samples prior to analysis because it is fast, simple, and inexpensive. It removes proteins from blood, plasma, and serum samples to reduce matrix interferences and prevent column clogging. A typical PPT protocol utilizes a 3:1 ratio of organic solvent to biological sample, which produces an extract containing approximately 75% organic solvent. In this presentation, we will examine the benefits and limitations of diluting protein-precipitated plasma prior to analysis versus injecting it directly. Performance will be assessed by comparing peak shapes, intensities, and recovery values for five antiretroviral (ARV) drugs (lamivudine, zidovudine, nevirapine, efavirenz, and ritonavir) in human plasma.
Methods: Calibration and quality control samples were prepared by fortifying human plasma with five ARV drugs and extracting by PPT. A portion of each sample extract was diluted 10x with mobile phase A (water + 0.01% acetic acid). All samples (10x diluted and undiluted extracts) were injected into a Shimadzu Nexera UHPLC equipped with a SCIEX API 4500 MS/MS. Detection was performed using electrospray ionization in positive ion mode with multiple reaction monitoring (MRM). Reversed-phase separations were performed using water and methanol mobile phases modified with 0.01% acetic acid under gradient conditions using a Restek Raptor Biphenyl 2.7 µm, 50 mm x 2.1 mm column equipped with a Raptor Biphenyl EXP 2.7 µm, 5 mm x 2.1 mm guard column.
Preliminary Data: Injections in solvents that are stronger than the mobile phase carry the sample through the column until the sample becomes fully diluted in mobile phase. This results in peak broadening or splitting, which negatively impacts quantitation, reproducibility, and sensitivity. The degree of peak distortion is a function of injection volume, column volume, and the difference in solvent strength between the injection solvent and the mobile phase. The smaller the column, the smaller the injection volume required to avoid strong solvent effects. By decreasing the injection volume, PPT extracts can be injected directly. Obvious differences in peak shape are observed for lamivudine, which elutes first in each chromatogram. When the size of the injection volume is reduced from 1 µL to 0.5 µL, uniform peak shape is achieved.
Calibration and quality control extracts were prepared and injected as described above to assess differences in recovery values for diluted and undiluted PPT extracts. Precision and accuracy values were comparable for each preparation across QC levels. Chromatograms from two separate injections of a 10 ng/mL calibration standard were compared. In one chromatogram, 0.5 µL of the undiluted standard extract is injected. In the other, the standard extract was diluted 10x in mobile phase A prior to injecting 5 µL. Both techniques result in the same amount of matrix and analyte on column with minimal differences in peak shape, intensity, or observed matrix interferences.
Lastly, column lifetime was evaluated by performing 500 injections of fortified undiluted PPT extract. It was noted that peak shapes remained consistent for all analytes throughout the experiment although a decrease in peak intensity was observed. Without additional experiments, it is difficult to conclude whether the loss in sensitivity was due to the direct injection of PPT extracts or another variable such as sample stability.
Novel Aspect: Direct injection of PPT extracts as an alternative to time-consuming solvent-exchange procedures or dilution without compromising the chromatography.
Small Molecules: Quantitative Analysis III, ThP 703
Analysis of Immunosuppressive Drugs from Whole Blood by LC-MS/MS
Susan Steinike (presenter), Justin Steimling, Frances Carroll
For more information, e-mail Susan Steinike.
Introduction: Immunosuppressive drugs are used to suppress the body’s immune response and are typically administered to prevent the rejection of transplanted organs or tissues. Cyclosporin A, tacrolimus, sirolimus, and everolimus are four of the most commonly used drugs in the therapy of organ transplantation. Due to their pharmacokinetic variabilities and narrow therapeutic indexes, time-sensitive and highly accurate therapeutic drug monitoring is necessary, not only to prevent rejection, but also to minimize toxic side effects. Therefore, a fast and accurate measurement of drug concentration in whole blood is critical to assist clinicians in the timely and proper treatment of patients. Herein, a high-throughput analysis combining a simple sample preparation step and a short chromatographic cycle time is discussed.
Methods: Human whole blood was fortified with four analytes to prepare the calibration standards and QC samples. For quantitation, cyclosporin D was used as the internal standard for cyclosporin A, and ascomycin was used as the internal standard for tacrolimus, sirolimus, and everolimus. The blood sample (100 mL) was mixed with 200 mL of precipitation solution (1:4 v/v 0.2M ZnSO4:methanol) containing 50 ng/mL of cyclosporin D and 5 ng/mL of ascomycin. The mixture was vortexed for 20 seconds at 3,000 rpm and then centrifuged for 10 minutes at 4,300 rpm. The supernatant was directly injected onto a Raptor Biphenyl column equipped with a Raptor Biphenyl EXP guard column for analysis using a Waters ACQUITY I-Class UPLC System coupled with a Xevo TQ-S mass spectrometer.
Preliminary Data: Linearity was evaluated in the range of 10-1,000 ng/mL for cyclosporin A and 1-100 ng/mL for tacrolimus, sirolimus, and everolimus. Using 1/x2 weighted linear regression for cyclosporin A and 1/x weighted linear regression for tacrolimus, sirolimus, and everolimus, all four compounds showed good linearity with r2 values of 0.999 or greater, and the % deviations were <10%. The signal-to-noise ratios of the lowest standard samples were from 100:1 to 300:1, indicating that this method could be used for the detection of much lower concentrations if necessary.
Three QC levels were prepared: 15, 150, and 800 ng/mL for cyclosporin A, and 5, 15, and 80 ng/mL for tacrolimus, sirolimus, and everolimus. Precision and accuracy analyses were performed on three different days. The method accuracy was demonstrated with %recovery <10% of the nominal concentration for all QC levels. The %RSD was 0.2-4.0% and 1.2-5.4% for intraday and interday, respectively, indicating good method precision. No chromatographic interferences were observed from the analysis of blank blood samples.
With a fast and simple sample preparation procedure and three minutes of chromatographic analysis time, the established method provides high-throughput therapeutic drug monitoring for these commonly used immunosuppressive drugs.
Novel Aspect: High-throughput therapeutic drug monitoring for commonly used immunosuppressive drugs in whole blood.