Sensitive and accurate characterization of Z drugs, metabolites and their degradation profile in urine using LC-MS/MS 

Using the SCIEX Triple Quad™ 5500+ System – QTRAP^® Ready and the SCIEX X500B QTOF System

Matthew Standland¹, Aaron Stella¹, and Xiang He^1
1SCIEX, USA

Introduction

Non-benzodiazepine sedative hypnotics, colloquially referred to as “Z drugs”, are a new class of sedative drugs that are structurally different than benzodiazepine but share similar mechanism of drug action. Consequently, Z drugs exhibit similar benefits such as hypnotic, sedative, and amnesiac effects through their pharmacological positive allosteric modulation of various subunits within the GABA receptor.¹ Although similar to those induced by benzodiazepines, their unique interaction at different subunits leads to different effects within the family of these drugs.

Z drugs are primarily indicated for the short-term treatment of insomnia. In addition to their sleep-inducing effects, this class of drugs present potential risks and adverse side effects such as nausea, drowsiness, dizziness, varying degrees of consciousness, and in extreme cases, coma and respiratory failure. The higher occurrence of these serious cases, some resulting in deaths, has prompted the FDA to impose stricter labeling requirements.² As a result, fast characterization of Z drug metabolism and secretion is critical due to the rapid onset of side effects. The ability to quickly identify and quantify commonly prescribed Z drugs and their metabolites in biological matrix is therefore paramount, especially in a case of poisoning or drug induced criminal behavior. 

In this technical note, a rapid, robust, and sensitive method for the detection and quantification of common Z drugs and their metabolites in human urine is described. Their degradation profile under different conditions was also investigated using both the SCIEX Triple Quad 5500+ System – QTRAP Ready and the SCIEX X500B QTOF System. The combination of the two systems enabled accurate and sensitive quantification of Z drugs and metabolites while providing confident identification of the degradation by-products.

Key features of LC-MS/MS method for accurate characterization of Z drugs in urine 

• Simple, dilute and shoot urine sample preparation procedure enabled rapid detection of Z drugs and their metabolites
• Robust data acquisition strategy using the SCIEX 5500+ System provided excellent linearity, accuracy and precision across the calibration range
• Degradation profile of Z drugs and their metabolites was investigated under several conditions
• Characterization of their degradation by-products was performed using both the SCIEX 5500+ System and SCIEX X500B QTOF System, which enabled analyte identification with high confidence through MS/MS spectral library matching
• Method is adaptable for full characterization of the degradation profile of other drugs and metabolites in biological matrix

Experimental details

Standards and internal standards: 5 Z drugs and 4 corresponding internal standards were procured from Cerilliant. Two solutions were prepared in methanol: a 10 µg/mL solution containing the Z drugs and a 1 µg/mL internal standard mixture containing the deuterated internal standards. The standard stock solutions were kept at -20°C. Figure 1 shows the structural diversity amongst Z drugs and includes 3 of the 5 Z drugs included in this panel (zolpidem, zopiclone and zaleplon). A list of the 5 Z drugs used in this study is provided in Table 1.

Calibrator preparation: The 10 µg/mL stock standard solution containing the 5 Z drugs targeted in this study was used to prepare a series of eleven calibrator solutions ranging from 1 to 2000 ng/mL. This was accomplished by spiking 100 µL of blank synthetic urine with the 10 µg/mL stock standard stock solution to reach the desired concentration levels, and then adding the 1 µg/mL internal standard mixture.

Sample preparation: A dilute and shoot sample preparation approach was used in this experiment. In brief, the spiked calibrator solutions were diluted 10 fold by diluting the 100 µL of blank synthetic urine with 10% methanol with 0.1% formic acid in water to a final volume of 1 mL. The resulting samples were then placed in an HPLC vial and then placed immediately in a chilled autosampler kept at 8 ºC.

Chromatography:  HPLC separation was performed on an ExionLC™ System using a Phenomenex Phenyl-Hexyl column (50 × 4.6 mm, 2.6 µm, 00B-4495-E0) held at 45ºC. Mobile phases used consisted of ammonium formate, methanol, and appropriate additives. The injection volume was 5 µL. The total LC runtime was 6 minutes when using the SCIEX 5500+ System and 12 minutes when using the SCIEX X500B QTOF System, respectively.

Mass spectrometry: Data were collected using two different instruments: a SCIEX 5500+ System for quantitative analysis and SCIEX X500B QTOF System for qualitative characterization of the degradation products of the Z drugs and their metabolites in urine. Both instruments were operated with electrospray ionization (ESI) in positive mode. The MRM algorithm was used on the SCIEX 5500+ System using Analyst^® Software. MS and MS/MS data were collected on the SCIEX X500B QTOF System using independent data acquisition (IDA). 10 maximum candidate ions with a minimum intensity threshold of 500 counts were selected for the IDA triggering criteria in SCIEX OS Software.

Data analysis: Data processing was performed using SCIEX OS Software. Quantitative analysis was performed in the Analytics module of the software where calibration curves, concentration calculations, assay precision and accuracy statistics were automatically generated. Positive analyte identification through MS/MS spectral library matching was performed using four main confidence criteria: mass error (M), retention time (R), isotope ratio difference (I), and library score (L).

Quantitative performance of the method using the SCIEX 5500+ System

Control urine samples spiked with the stock standard solution and the internal standard mixture were prepared using the aforementioned dilute and shoot sample preparation procedure. Three replicates of each sample were injected to build a data analysis processing method.

Figure 2 shows the extracted ion chromatogram (XIC) for the 5 Z drugs and 4 corresponding internal standards. Separation of the analytes was accomplished using a 5 minute LC runtime, which was sufficient to achieve near baseline separation of all the Z drugs in the panel.

The quantitative performance of the method was investigated by injecting the series of calibrator samples spiked with various concentrations ranging from 1 to 2000 ng/mL in blank synthetic urine. Calibration curves were generated to evaluate the sensitivity and linearity of the method. Figure 3 shows the regression curves for each of the 5 Z drugs. The series of nine calibrator solutions displayed excellent linearity over the calibration range, with R² values greater than 0.99 for each of the five analytes.

The series of calibrator solutions was also used to determine the precision and accuracy of the method at the low end of the concentration range. Table 1 lists the precision of measurement at the three lowest calibrator levels (1, 2.5 and 5 ng/mL) for each of the 5 Z drugs targeted in this study. The %CV values ranged from 0.75 to 11.65%, demonstrating the robustness of the method at the low end of the calibration range. Accuracies for the spiked urine samples ranged from 80 to 100% (data not shown). Overall, the method showed excellent robustness and quantification performance, as demonstrated by the excellent reproducibility and linearity observed across the calibration range.  

Characterization of the Z drugs degradation profile using a combination of the SCIEX 5500+ System and the X500B QTOF System 

Urine is a common biological matrix used for detection of Z drugs, and the favored matrix for cases involving drug-facilitated crimes where the detection window is longer than other matrices, such as blood or plasma.³ Since both the onset and metabolism of these drugs is fast, it is critical to be able to quickly and accurately quantify Z drugs and their metabolites before degradation occurs.⁴ Out of the 5 Z drugs studied, zopiclone is reported to exhibit rapid degradation in matrix, even when stored under refrigeration.^{5, 6} This analyte is also known to be especially susceptible to degradation in basic environment, resulting in the breakdown conversion to 2-amino-5-chloropyridine (ACP).⁴ 

The degradation profile of zopiclone was investigated under various conditions over time and characterized using both the SCIEX 5500+ System and the SCIEX X500B QTOF System. Zopiclone spiked in synthetic urine was subjected to five different solvent conditions over the course of 840 minutes in a chilled HPLC autosampler kept at 8 ºC. Figure 4 shows the degradation profile of zopiclone over time under 5 various conditions. The traces shown on the plot represent the peak area of zopiclone as a function of exposure time. Degradation of zopiclone is observed under neutral and weakly basic conditions (10% methanol, 10% methanol with 0.1% ammonium bicarbonate, pH 7.5 and 10% methanol with 0.1% ammonium hydroxide), as evidenced by the peak area decrease. However, zopiclone appears to be stable under acidic conditions (10% methanol with 0.1% formic acid and 10% methanol with 0.1% acetic acid). These results suggest that the rapid degradation of zopiclone can be alleviated with addition of acid. 

The degradation profile of zopiclone and conversion to ACP was further investigated using the acquired TOF-MS/MS spectra on the SCIEX X500B QTOF System. Figure 5 shows the XIC, TOF MS and TOF MS/MS spectra of ACP detected in a synthetic urine sample containing 1 ng/mL of zopiclone under basic condition. IDA generated a comprehensive and high-quality MS/MS spectra, enabling accurate and confident identification of ACP as a degradation by-product of zopiclone in urine by using library spectral matching. SCIEX OS Software confirmed identification of ACP using mass error, retention time, isotopic distribution, and MS/MS spectral library matching. The results highlight the use of the X500B QTOF System as a powerful tool for reliable and confident compound identification through MS/MS spectral library matching.

Conclusions

A sensitive and robust method was developed for the accurate quantification and characterization of 5 Z drugs and their metabolites in human urine. The quantitative performance of the method was investigated using the SCIEX 5500+ System. The method provided excellent linearity, accuracy and precision across concentrations ranging from 1 to 2000 ng/mL. The degradation profile of one of the Z drugs, zopiclone, was investigated using the SCIEX X500B QTOF System. The results showed that zopiclone was prone to degradation under neutral and weakly basic conditions. The combination of IDA for data acquisition and the use of confidence criteria enabled accurate identification of ACP, one of the degradation by-products of zopiclone in urine. Overall, the results show that the combined use of the SCIEX 5500+ System and the SCIEX X500B QTOF System enables full qualitative and quantitative analysis of Z drugs, metabolites and degradation by-products with a high level of sensitivity, specificity, accuracy and precision.  

References

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2. Taking Z-drugs for Insomnia? Know the Risks. FDA 04/30/2019.
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