A Rapid LC-MS-MS Method for the Quantitation of Antiepileptic Drugs in Urine.

Epilepsy is a common neurologic disease that requires treatment with one or more medications. Due to the polypharmaceutical treatments, potential side effects, and drug-drug interactions associated with these medications, therapeutic drug monitoring is important. Therapeutic drug monitoring is typically performed in blood due to established clinical ranges. While blood provides the benefit of determining clinical ranges, urine requires a less invasive collection method, which is attractive for medication monitoring. As urine does not typically have established clinical ranges, it has not become a preferred specimen for monitoring medication adherence. Thus, large urine clinical data sets are rarely published, making method development that addresses reasonable concentration ranges difficult. An initial method developed and validated in-house utilized a universal analytical range of 50-5,000 ng/mL for all antiepileptic drugs and metabolites of interest in this work, namely carbamazepine, carbamazepine-10,11-epoxide, eslicarbazepine, lamotrigine, levetiracetam, oxcarbazepine, phenytoin, 4-hydroxyphenytoin, and topiramate. This upper limit of the analytical range was too low leading to a repeat rate of 11.59% due to concentrations >5,000 ng/mL. Therefore, a new, fast liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with a run time under 4 minutes was developed and validated for the simultaneous quantification of the previously mentioned nine antiepileptic drugs and their metabolites. Urine samples were prepared by solid-phase extraction and analyzed using a Phenomenex Phenyl-Hexyl column with an Agilent 6460 LC-MS-MS instrument system. During method development and validation, the analytical range was optimized for each drug to reduce repeat analysis due to concentrations above the linear range and for carryover. This reduced the average daily repeat rate for antiepileptic testing from 11.59% to 4.82%. After validation, this method was used to test and analyze patient specimens over the course of approximately one year. The resulting concentration data were curated to eliminate specimens that could indicate an individual was noncompliant with their therapy (i.e., positive for illicit drugs) and yielded between 20 and 1,700 concentration points from the patient specimens, depending on the analyte. The resulting raw quantitative urine data set is presented as preliminary reference ranges to assist with interpreting urine drug concentrations for the nine aforementioned antiepileptic medications and metabolites.

Endogenous GHB in Segmented Hair Part II: Intra-individual Variation for Exogenous Discrimination.

The endogenous presence of gamma-hydroxybutyric acid (GHB) complicates the interpretation of results in cases where an exogenous dosing is suspected. Due to GHB's rapid metabolism and clearance following exogenous doses, hair has become a preferential matrix for confirmation of GHB exposure in drug-facilitated crimes. However, unlike blood and urine where an agreed-upon cut-off concentration for differentiation between endogenous and exogenous GHB has been made, there has been no consensus on a cut-off concentration for hair. This is due in part to the wide inter- and intra-individual variation that has been observed in endogenous GHB hair studies. A large (>50) population study of 214 donors was conducted to better understand these variations and to evaluate whether a cut-off concentration could be established for endogenous GHB in human hair. As seen in our previous study, the inter-individual variation was large, with concentrations ranging from <0.40 to 5.47 ng/mg. This range made an absolute cut-off concentration recommendation inappropriate, so an alternative approach for GHB discrimination was investigated utilizing the intra-individual variation. Male donors appeared to have greater intra-individual variation than female donors, yet it was noted that segment-to-segment variation along the length of hair had minimal change between individual donor's adjacent segments. Overall, 97.1% of the adjacent segment differences were within ±0.5 ng/mg. Therefore, instead of a recommended cut-off concentration, it appears that using adjacent segment concentration differences could be a strategy to assist in differentiating endogenous from single exogenous GHB exposure. In the absence of controlled dosing data, previously published segmented results from controlled and suspected dosing donors are examined using the adjacent segmental difference approach and the results compared to currently used ratio-based calculations.

Endogenous GHB in Segmented Hair Part I: Inter-individual Variation for Group Comparisons.

While earlier studies have attempted to resolve the challenges encountered when interpreting gamma-hydroxybutyric acid (GHB) concentrations in hair (primarily due to its endogenous presence), few have had large sample sizes. The first objective of this study was to evaluate the inter-individual variation of endogenous GHB concentrations. The second objective, to be detailed in another report, was to assess intra-individual variation and the impact on exogenous GHB discrimination. Over 2,000 hair segments from 141 women and 73 men (all processed hair 3-12 cm long) were analyzed in this study. The raw calculated range of endogenous GHB concentrations was <0.40-5.47 ng/mg with 97.5% of the segmental results calculated less than 2.00 ng/mg. Imputation, assuming a lognormal distribution, was applied to the data to include non-detect (ND) data (

A Dilute and Shoot LC-MS/MS Method for Antipsychotics in Urine.

Adherence to prescribed antipsychotics is an ongoing problem. Traditionally, estimates of adherence have been made from patient interviews, pill counting and blood testing. A number of methods for the analysis of antipsychotics in blood have been reported for both therapeutic drug monitoring and postmortem testing for toxicity. This report details a dilute and shoot method for the analysis of 19 different antipsychotics and metabolites. The method takes advantage of earlier reports demonstrating unique, prevalent urine metabolites for aripiprazole, brexpiprazole, haloperidol and lurasidone to enhance sensitivity for these analytes. With a fast analysis time and minimal sample preparation, this method can be used for quantitation of antipsychotics in urine. Finally, this method has been used to test samples for over a year with the results summarized in this report. While further improvements are certainly possible, this method is selective and sensitive for this group of important compounds.

Development and Validation of a Novel All-Inclusive LC-MS-MS Designer Drug Method.

Designer drugs including synthetic cannabinoids and synthetic cathinones are an increasing problem due to the ease of access to these compounds. They present analytical challenges inasmuch as the compound structures are numerous and growing within each class. Typically each class of designer compounds is analyzed separately due to differences in chemistry, desired cut-offs or other reasons. Physicians treating "high-risk" patients typically order tests for all "illicit" substances which can span several test classes. Despite that multiple classes of designer drugs are ordered together, there has not been a comprehensive confirmatory test developed to date. Presented here is a novel comprehensive designer drug LC-MS-MS method that combines synthetic cannabinoids and synthetic cathinones, etizolam, a designer benzodiazepine and mitragynine (kratom), a natural product analgesic. This method improves laboratory throughput with a cycle time of ~4.5 min which affords resolution of crucial isomers, such as ethylone and butylone. Development of this method also provided an opportunity to update the list of compounds within the method. Analytes with fewer than five positive specimens in a year of testing with previous separate methods were removed as old and not current. New analytes were added based on reports from NMS Laboratories and the US Drug Enforcement Administration testing and drug seizures, which included etizolam, its major metabolite α-hydroxyetizolam as well as newer synthetic cannabinoids (5-fluoro ADB metabolite 7, AB-FUBINACA metabolite 3, AB-FUBINACA metabolite 4 and MDMB-FUBINACA metabolite M1) and synthetic cathinones (N-ethyl pentylone). Finally, the impact of the new analytes and cut-off changes are discussed in context with patient results from the first 4 months of testing after implementation of the method in the lab.

Impact of ß-Glucuronidase Mediated Hydrolysis on Haldol® Urinalysis.

Reports have suggested that patients with mental health disorders including major depressive disorder and schizophrenia have dramatically low adherence levels to prescribed medications. Patients on haloperidol (Haldol®) therapy, regardless of their disease, were found to have higher adherence levels-though still strikingly low. This work shows that high levels of the glucuronidated form of haloperidol are present in patient urine samples. Time-of-Flight (TOF) mass spectrometry experiments are consistent with both the presence of haloperidol glucuronide and that hydrolysis of haloperidol patient urine samples leads to significantly increased concentrations of free haloperidol. Urine samples collected from patients prescribed haloperidol were tested with and without hydrolysis revealing a significant increase in the number of patients testing positive when the samples were hydrolyzed before analysis. These data demonstrate that hydrolysis greatly improves the sensitivity and consistency of results for patients on haloperidol therapy resulting in positivity data that strongly correlates with the dosage form administered.

Analytical Considerations When Developing an LC-MS/MS Method for More than 30 Analytes.

BACKGROUND: While validation of analytical (LC-MS/MS) methods has been documented in any number of articles and reference texts, the optimal design and subsequent validation of a method for over 30 analytes presents special challenges. Conventional approaches to calibration curves, controls, and run time are not tenable in such methods. This report details the practical aspects of designing and implementing such a method in accordance with College of American Pathologists validation criteria. METHODS: Conventional criteria were followed in the design and validation of a method for 34 analytes and 15 internal standards by LC-MS/MS. These criteria are laid out in a standard operating procedure, which is followed without exception and is consistent with College of American Pathologists criteria. RESULTS: The method presented herein provides quality results and accurate medication monitoring. The method was optimized to negate interferences (both from within the method and from potential concomitant compounds), increase throughput, and provide reproducible quality quantification over relevant analyte concentrations ranges. CONCLUSIONS: The method was designed primarily with quality and accurate medication monitoring in mind. The method achieves these goals by use of novel approaches to calibration curves and controls that both improve performance and minimize risk (financial and operational). As automation and LC-MS/MS equipment continue to improve, it is expected that more methods like this one will be developed.

Quetiapine Carboxylic Acid and Quetiapine Sulfoxide Prevalence in Patient Urine.

Treatment adherence is often an issue with mental health patients. For those prescribed quetiapine (Seroquel®), the low levels of parent drug and plasma metabolite(s) (e.g., 7-hydroxyquetiapine) typically used in urine drug monitoring can result in false negatives with concomitant unfavorable impacts on patient care. Literature review coupled with liquid chromatography/time-of-flight mass spectrometry analysis of patient positive urine samples indicated the presence of quetiapine carboxylic acid and quetiapine sulfoxide as significant urinary metabolites of quetiapine. Analysis of these two metabolites determined that they are abundant in the urine of quetiapine patients and can result in apparent adherence rates that are improved relative to those determined using only quetiapine and 7-hydroxyquetiapine. For example, analysis of a random set of 114 patients who were prescribed quetiapine exhibited an apparent adherence rate of 47% using the quetiapine carboxylic acid and quetiapine sulfoxide metabolites. Traditional metabolite testing with quetiapine and 7-hydroxyquetiapine yielded apparent adherence rates of ~31% while all four analytes resulted in apparent adherence of 48%. The prevalence of these metabolites suggests that quetiapine urine drug testing would be more consistent with prescriptions when they are included in the analysis.

Capillary electrophoresis-mass spectrometry determination of morphine and its isobaric glucuronide metabolites.

The determination of morphine and its isobaric metabolites morphine-3-beta-d-glucuronide (M3G) and morphine-6-beta-d-glucuronide (M6G) is useful for therapeutic drug monitoring and forensic identification of drug use. In particular, capillary electrophoresis with mass spectrometry (CE-MS) represents an attractive tool for opioid analysis. Whereas volatile background electrolytes in CE often improve electrospray ionization for coupled MS detection, such electrolytes may reduce CE separation efficiency and resolution. To better understand the effects of background electrolyte (BGE) composition on separation efficiency and detection sensitivity, this work compares and contrasts method development for both volatile (ammonium formate and acetate) and nonvolatile (ammonium phosphate and borate) buffers. Peak efficiencies and migration times for morphine and morphine metabolites were optimal with a 25mM ammonium borate buffer (pH=9.5) although greater sensitivities were achieved in the ammonium formate buffer. Optimized CE methods allowed for the resolution of the isobaric morphine metabolites prior to high mass accuracy, electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS detection applicable to the analysis of urine samples in under seven minutes. Urine sample preparation required only a 10-fold dilution with BGE prior to analysis. Limits of detection (LOD) in normal human urine were found to be 1.0µg/mL for morphine and 2.5µg/mL for each of M3G and M6G by CE-ESI-QTOF-MS. These LODs were comparable to those for CE-UV analysis of opioid standards in buffer, whereas CE-ESI-QTOF-MS analysis of opioid standards in buffer yielded LODs an order of magnitude lower. Patient urine samples (N=12) were analyzed by this new CE-ESI-QTOF-MS method and no significant difference in total morphine content relative to prior liquid chromatography-mass spectrometry (LC-MS) results was found as per a paired-t test at the 99% confidence level. Whereas the LC-MS method applied to these samples determined only total morphine content, this new CE-ESI-QTOF-MS method allowed for species differentiation in addition to total morphine determination. By this method, it was found that M3G and M6G metabolites were present in a 5:1 concentration ratio, on average, in patient samples. Therefore, the CE-ESI-QTOF-MS method not only allows for total morphine concentration determination comparable to established LC-MS methods, but also allows for differentiation between morphine and its trace glucuronides, yielding additional biochemical information about drug metabolism.

Rapid enzymatic hydrolysis using a novel recombinant ß-glucuronidase in benzodiazepine urinalysis.

Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant ß-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant ß-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone ß-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant ß-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant ß-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation.

Thermodynamic analysis of protein folding and stability using a tryptophan modification protocol.

Described here is the development of a mass spectrometry-based covalent labeling protocol that utilizes the reaction of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNSB) with tryptophan (Trp) residues to measure protein folding free energies (ΔG(f) values). In the protocol, the chemical denaturant dependence of the rate at which globally protected Trp residues in a protein react with HNSB is evaluated using either a matrix assisted laser desorption ionization time-of-flight analysis of the intact protein or a quantitative, bottom-up proteomics analysis using isobaric mass tags. In the proof-of-principle studies performed here, the protocol yielded accurate ΔG(f) values for the two-state folding proteins, lysozyme and cytochrome c. The protocol also yielded an accurate measure of the dissociation constant (K(d) value) for the binding of N,N',N″-triacetylchitotriose to lysozyme, and it successfully detected the binding of brinzolamide to BCA II, a non-two-state folding protein. The HNSB protocol can be used in combination with SPROX (stability of proteins from rates of oxidation), a previously reported technique that exploits the hydrogen peroxide oxidation of methionine (Met) residues in proteins to make ΔG(f) value measurements. Incorporating the HNSB protocol into SPROX increased the peptide and protein coverage in proteome-wide SPROX experiments by 50% and 25%, respectively. As part of this work, the precision of proteome-wide ΔG(f) value measurements using the combined HNSB and SPROX protocol is also evaluated.

False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform.

Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2% and <0.8% are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.

Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, ß-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.