Factors Compromising Glucuronidase Performance in Urine Drug Testing Potentially Resulting in False Negatives.

Next generation ß-glucuronidases can effectively cleave glucuronides in urine at room temperature. However, during the discovery studies, additional challenges were identified for urine drug testing across biologically relevant pH extremes and patient urine specimens. Different enzymes were evaluated across clinical urine specimens and commercially available urine control matrices. Each enzyme shows distinct substrate preferences, pH optima, and variability across clinical specimens. These results demonstrate how reliance on a single glucuronidated substrate as the internal hydrolysis control cannot ensure performance across a broader panel of analytes. Moreover, sample specific urine properties compromise ß-glucuronidases to varying levels, more pronounced for some enzymes, and thereby lower the recovery of some drug analytes in an enzyme-specific manner. A minimum of 3-fold dilution of urine with buffer yields measurable improvements in achieving target pH and reducing the impact of endogenous compounds on enzyme performance. After subjecting the enzymes to pH extremes and compromising chemicals, one particular ß-glucuronidase was identified that addressed many of these challenges and greatly lower the risk of failed hydrolyses. In summary, we present strategies to evaluate glucuronidases that aid in higher accuracy urine drug tests with lower potential for false negatives.

Norbuprenorphine Interferences in Urine Drug Testing LC-MS-MS Confirmation Methods from Quetiapine Metabolites.

Norbuprenorphine interferences were observed in urine drug testing LC-MS-MS confirmation methods used to assess patient compliance with prescribed buprenorphine for chronic pain and opioid use disorder. The interferences were observed in the norbuprenorphine MS-MS transitions, m/z 414.4/83.1 and 414.4/187.2, at and near the norbuprenorphine retention time at multiple laboratories using different sample preparation procedures and chromatographic conditions. When the interferences were present, a norbuprenorphine result could not be reported. Upon investigation, the interferences were correlated with prescribed quetiapine (Seroquel, Seroquel XR), a second-generation antipsychotic medication approved for the treatment of schizophrenia, bipolar disorder and more recently as an adjunct treatment for major depressive disorder. In addition to the approved indications, quetiapine is prescribed off-label for other conditions including insomnia and anxiety disorders. Off-label prescribing has increased in recent years, thereby exacerbating this analytical issue. Here, we present the study of four quetiapine metabolites found to have significant direct or potential interferences in norbuprenorphine quantitation. The four metabolites were putatively identified as two hydroxyquetiapine acids differing in the site of hydroxylation and a quetiapine sulfoxide acid diastereomer pair. As a result of this study, interference-free norbuprenorphine MS-MS transitions, m/z 414.4/340.2 and 414.4/326.1, were found that were selective for norbuprenorphine while maintaining an acceptable 10 ng/mL lower limit of quantitation.

Characterization of an Amphetamine Interference from Gabapentin in an LC-HRMS Method.

An amphetamine interference was observed during the development of an liquid chromatography-high-resolution mass spectrometry (LC-HRMS) multi-class confirmation method for the determination of 47 drugs and metabolites in urine. The interference passed all qualitative criteria for amphetamine leading to potential false-positive results. Upon investigation, it was found that the amphetamine interference was correlated with the presence of high levels of gabapentin. Gabapentin is routinely detected in patient urine specimens at levels in excess of 1 mg/mL as it is widely prescribed at high doses and does not undergo significant metabolism. The source of the interference was identified as a gabapentin in-source fragment isomeric with protonated amphetamine. Here we describe the characterization of this interference and how its effect was mitigated in the LC-HRMS method.

Analysis of isomeric opioids in urine using LC-TIMS-TOF MS.

In the present work, a fast separation, identification and quantification workflow based on liquid chromatography coupled to trapped ion mobility in tandem with mass spectrometry (LC-TIMS-MS) is described for the analysis of common isomeric drugs of abuse and their metabolites in human urine. In particular, the analytical performance of LC-TIMS-MS is shown for identification based on retention time, collision cross section and accurate mass for three sets of common isomeric opioids and their deuterated analogs in urine. The LC-TIMS-MS analysis provided limits of detection of 1.4-35.2 ng/mL with demonstrated linearity up to 500 ng/mL, enabling discovery and targeted monitoring (DTM) of opioids in urine, with high precision in retention times (RT) (< 0.3%), collision cross sections (CCS) (< 0.6%) and mass accuracy (< 1 ppm) across multiple measurements using external calibration. A good agreement was observed between theoretical and experimental CCS from candidate structures optimized at the DFT/B3LYP level. The need for complementary liquid and mobility separations prior to mass analysis is shown for the analysis of complex mixtures, with mobility resolving power of 80-130. The reproducibility and high speed of LC-TIMS-MS analysis provides a powerful platform for drug and metabolite screening in biological matrices with higher precision and confidence than traditional LC-multiple reaction monitoring (MRM) approaches.

Identification of Novel Opioid Interferences using High-Resolution Mass Spectrometry.

Novel opioid interferences were observed during the development of a high-resolution liquid chromatography-mass spectrometry urine drug testing method for 47 analytes from multiple drug classes. The interferences affected both analytes and internal standards and were only observed when the method was challenged with patient samples. Some interferences were attributable to isomeric opioid metabolites not previously reported while others were due to interference from in-source dissociations or 13C isotopic contributions from known opioid metabolites not typically monitored as analytes. Based on patient drug profiles, known and inferred metabolism, accurate mass, retention time and MS/MS spectrum, the putative identity of each interference was assigned and later confirmed, when possible, using an authentic standard. Opioids are some of the most frequently monitored analytes in urine drug testing laboratories. Because of the potential for co-purification, co-chromatography and spectral similarity, it is anticipated that the reported opioid metabolite interferences could be present with other method conditions and instrument platforms. The objectives of this work are to raise awareness of these interferences and emphasize the importance of evaluating patient samples for potential interferences during method development.

Development and validation of a high performance liquid chromatography-diode array detection method for the determination of aging markers in tequila.

A solid-phase extraction procedure followed by analysis by high performance liquid chromatography (HPLC) with UV-vis photodiode array detection (DAD) is proposed to simultaneously determine 11 aging markers in tequila. The method showed good intraday (n=5) and interday (n=3) precision, RSD<1.6% in both cases, for each of the identified compounds. The calibration curves were linear at the tested ranges (R(2)>0.999). Good recoveries (84.2-108.5%) were obtained for 10 of the 11 compounds studied; and the LOD and LOQ ranged from 0.62 to 4.09 microg/mL and 1.9-12.4 microg/mL, respectively. The proposed methodology was applied to a set of 15 authentic tequila samples grouped by aging state (blanco, reposado and añejo). An ANOVA analysis combined with discriminant analysis with stepwise backward variable selection was used to differentiate between the various aging groups based on their oak related compounds content.

Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures.

We demonstrate the effectiveness of laser-induced fluorescence (LIF) for monitoring the development and stress detection of in vitro tissue cultures in a nondestructive and noninvasive way. The changes in LIF spectra caused by the induction of organogenesis, the increase of the F690/F740 ratio as a result of the stress originated in the organogenic explants due to shoot emergence, and the relationship between fluorescence spectra and shoot development were detected by LIF through closed containers of Saintpaulia ionantha.