Oneka T. Cummings

A novel "dilute-and-shoot" method is described for the analysis of "bath salts" sold as "legal" highs, including mitragynine and nine synthetic cathinones. The cycle time for this method is 2.5 min. No sample clean-up or extraction was performed. This assay monitors two tandem mass spectrometry (MS/MS) transitions for each of the following 10 analytes: alpha-PVP, butylone, ethylone, MDPV, mephedrone, methcathinone, methedrone, methylone, naphyrone, and mitragynine; and two internal standards: methylone D3 and alpha-PVP D8. This straightforward "dilute-and-shoot" liquid chromatography (LC)–MS/MS method produces acceptable limit of detection (LOD) and limit of quantitation (LOQ) limits without costly extraction and time-consuming concentration protocols.

	Cathinone is a monoamine alkaloid found in the Khat plant (

 (1). Khat is a shrub grown in eastern Africa and the southern Arabian Peninsula, which can have mild stimulant effects when the leaves are chewed (2). Cathinone is also a beta ketone analog of amphetamine and as such is reported to cause sympathomimetic effects as well as psychoactive effects common with amphetamine and amphetamine derivatives such as MDMA ("Ecstasy") and MDA ("Eve") (3). Chewing of the Khat plant has been linked to increased risk of heart-related diseases and duodenal ulcers (4). Synthetic variants of cathinone, commonly referred to as "bath salts" or as "novel psychoactive substances" (NPS) by public health personnel, are stronger and more dangerous than their natural counterpart (5). The first synthetic derivatives of cathinone were reported during the 1920s (6-8). These initial derivatives were designed for medical purposes; however, most were withdrawn as a result of abuse and obvious dependency among users. Interestingly, bupropion (Wellbutrin) is a "synthetic cathinone" and is widely used as an antidepressant and anti-anxiety medicine (2).

	More recently, synthetic cathinones (NPS) have expanded as popular drugs of abuse. Their ready availability in the local drug market and so-called head shops make them easy "legal" highs. These active agents are sold under various names such as pesticides and insect repellants. They are often labeled as "not for human consumption," to avoid regulatory control. However, the use of bath salts has significantly increased in recent years with poison control centers in the United States reporting 304 calls related to bath salts in 2010 and 1782 calls in the first four months of 2011 (9,10). It is believed that the increase in their use is a result of their availability, lack of proper methods for drug testing, and media coverage.

	Mitragynine is a naturally occurring substance found in the leaves of the kratom (

) plant, a member of the coffee family found in Southeast Asia, Indochina, and Malaysia (11). This "legal" high is readily available and is often used to mitigate pain, aid in opiate withdrawal, and for recreational purposes (12). This natural drug is regulated in a number of countries including Malaysia but not currently scheduled in the United States (13).

	Urine drug testing is a common means of monitoring for the use of these drugs. While forensic laboratories often have access to blood, urine, and other tissues to test in post-mortem subjects, pain management laboratories tend to focus on urine or oral fluids because of the ease of sampling and minimal stability requirements. As well as ease of sample collection, the sample preparation required is comparatively easier for urine than other patient specimens, for example, blood. Often such preparation consists of sample matrix removal using solid-phase extraction (SPE); however, reports of “dilute-and-shoot” testing confirm that direct analysis without sample preparation is possible for some analytes (14). In this study, a unique “dilute-and-shoot” liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the analysis of nine synthetic cathinones and mitragynine (Kratom) in patient urine samples. This method is ideal for a high-throughput production environment because it has a modest 2.5-min cycle time and requires low sample preparation time and cost while still preserving sensitivity and specificity of the quantitative analytical result. Above all, this method achieved complete chromatographic resolution of the isobars, ethylone and butylone, both of which are analyzed in this method, by lowering column temperature to 20 °C and cutting flow rate to 0.4 mL/min to maintain system pressure.

	All reference standard compounds, including internal standards, were purchased from Cerilliant. Solvents, including methanol, acetonitrile, and formic acid, were purchased from VWR. Normal drug-free urine was obtained from UTAK.

	Aliquots of normal drug-free urine were fortified with the reference standards at concentrations of 25 ng/mL, 250 ng/mL, and 1000 ng/mL as calibrators. All samples, calibrators, and controls were diluted 5× with the internal standard solution (0.5 µg/mL methylone D3 and alpha-PVP D8) by adding 400 µL of internal standard to 100 µL of sample.

	All analyses were performed via LC–MS/MS on Acquity TQD UPLC/MS/MS system (Waters). A 2.1 × 50 mm, 1.7-µm Acquity UPLC BEH C18 analytical column (Waters) was used for chromatographic separations. The run time for this method was 2.0 min with a total cycle time of 2.5 min. No sample extraction or clean-up was required before LC–MS/MS analysis. Two transitions were monitored for each of 10 analytes (alpha-PVP, butylone, ethylone, MDPV, mephedrone, d,l-methcathinone, methedrone, methylone, mitragynine, and naphyrone), plus two internal standards (methylone D3 and alpha-PVP D8), as shown in Table I. Elution solvents used in the gradient were 0.1% formic acid in 10% methanol (mobile phase A) and 0.1% formic acid in 50:50 methanol– acetonitrile (mobile phase B), with a flow rate of 0.4 mL/min (Table II).

	The method was validated by examining various parameters such as limits, linearity, precision and accuracy (P&A: Ten replicates of each of three concentrations each day for three days with coefficient of variance [%CV] of each set of P&A point replicates each day and across all P&A days falling within +/- 15%), interferences, and matrix effects. Drugs of abuse (benzoylecgonine, THC-A, amphetamine, methamphetamine, MDMA, MDEA, MDA, and phentermine) and therapeutic drugs (oxazepam, morphine, imipramine, buprenorphine, fentanyl, meprobamate, methadone, tramadol, gabapentin, pregabalin, and tapentadol) were tested as possible sources of interferences. Patient samples were evaluated by comparing the quantitative results of a previously used method with those of the new method as described herein. Since mitragynine and alpha-PVP were not evaluated in the previous method, a comparison was not possible. The previous method was developed and validated as described here but lacked critical analytes, for example, mitragynine and alpha-PVP. This method has not been reported in the literature. Detailed discussion of these validation parameters can be found in a paper by Enders and McIntire (14).

	The tolerance for all analytes at each concentration level was ± 25%. All daily calibration curves had correlation coefficient values (r

) greater than or equal to 0.99. The matrix blanks following the highest calibrator points did not show any signs of carryover, that is, no response greater than 50% of the limit of quantitation (LOQ).


	Figure 1: Overlaid chromatogram of 10 analytes determined in this assay. The chromatogram was obtained after injecting a sample at the limit of quantification (LOQ) of 25 ng/mL. Internal standards (IS) are not shown.

	Two mass transitions were selected for each of the analytes following infusion studies of pure standards dissolved in matrix. The first transition served as the quantifier ion transition and the second transition served as a qualifier ion transition (Table I). The ratio of the areas of the quantifier ion transitions for the analyte of interest and the internal standard (IS) were used to obtain the relative response by plotting the area ratio of the analyte-to-IS versus analyte concentration. The area of the second ion transition of the analyte was used to generate a specific relative ratio to the area of the quantifier peak. This generates a compound specific “ion ratio” for each analyte that together with retention time helps to confirm that the peak results from the compound of interest.


	Figure 2: Chromatogram showing the separation of the isomers ethylone and butylone. 

	This method generated calibration data with consistent peak areas and ion ratios for all analytes and internal standards. Daily three-point calibration curves were completed with correlation coefficient values (r

) of at least 0.99 for all analytes. Consistent accuracies of 100 ± 25% were obtained for all calibration points, negative and positive controls, as well as points generated daily for both calibration and controls. The limit of quantification (LOQ) was determined to be 25 ng/mL, which was sufficient to achieve consistent precision and accuracies as well as acceptable ion ratios, while the upper limits of linearity and of carryover (ULOL, ULOC) were 1000 ng/mL. The chromatogram of all analytes shown in Figure 1 provides clear evidence that there is good chromatographic separation and resolution of all analytes assessed in this assay even at the LOQ. Furthermore, Figure 2 shows the separation achieved for the ethylone and butylone isobars, while Table III shows a summary of the validation results.


	Table IV: Positive NPS data from a 14-month period of testing

	This work documents the successful development and validation of an improved LC–MS/MS method for routine, high-throughput analysis of alpha-PVP, butylone, ethylone, MDPV, mephedrone, d,l-methcathinone, methedrone, methylone, mitragynine, and naphyrone, using methylone D3 and alpha-PVP D8 internal standards. During the initial stages of method development, the column temperature was held at 50 °C with a flow rate of 0.8 mL/min, which required a much longer 4.0 min cycle time for complete resolution of all analytes of interest. This was especially true for the isobars butylone and ethylone, which did not exhibit robust separation even with the longer elution time. To ensure complete resolution (< 10% peak height overlap) of these two isobars while still achieving the desired 2.5 min cycle time, the column temperature was lowered to 20 °C and the flow rate was decreased to 0.4 mL/min. Traditional chromatography paradigms indicate that higher temperatures lead to better resolution (15). However, the unique combination of column, solvents, analytes, and flow rates used here responded to lowering the temperature for improved resolution. Lowering the column temperature coupled with decreasing the gradient flow rates proved vital to ensure that appropriate system pressures were maintained while facilitating the appropriate degree of separation of the isomers, ethylone and butylone.

	The synthetic cathinone compounds of interest were successfully analyzed in urine samples by this “dilute-and-shoot” LC–MS/MS method. This particular method is robust and useful because it reduces analysis time and does not incur additional costs for up-front sample preparation while still providing accurate results. As there were no interferences observed from the major drugs of abuse and therapeutic drugs, this method is an excellent tool for the clinical, medicinal, and toxicology communities, especially in high sample volume settings.

	Results achieved with this method are summarized in Table IV where positive data from over a year of testing are summarized. Mitragynine was the most prevalent component in the tested urine samples, while alpha-PVP and methcathinone were close seconds. Although tests for these analytes are positive for just a small fraction of all pain medication monitoring samples, these results are important to both physicians and patients in their ongoing treatments for chronic pain, substance abuse, and mental illness. For example, patient ages associated with at least one positive bath salt result ranged from 19 to 72 years old, with equal positivity rates across all age groups. Lastly, the overwhelming positivity of mitragynine in this group more than likely reflects the legality of this natural product in many states in the USA, while attempts to schedule this agent at the federal level have been delayed by consumer activist protests (13).

	Any method that tests for illicit synthetic substances must be updated frequently to keep up with the changes on “the street” that result in novel pharmaceutical substances being consumed with known and unknown effects. However, this method offers advantages in terms of cost and time and has been shown to be capable of detecting many patient positives when implemented for large numbers of samples.

	The authors would like to thank the National Science Foundation (NSF-GOALI Award 1611072) for financial support of this work, together with Wake Forest University and Ameritox.

	(4) A. Al-Motarreb, M. Al-Habori, and K.J. Broadley, 

	(6) J.F. Hyde, E. Browning, and R. Adams, 

	(7) C.D. Rosenbaum, S.P. Carreiro, and K.M. Babu, 

	(8) C.L. German, A.E. Fleckenstein, and G.R. Hanson, 

https://www.medicinenet.com/bath_salts_abuse_and_addiction/article.htm

	(11) W.C. Prozialeck, J.K. Jivan, and S.V. Andurkar,

	(12) M.A. Rech, E. Donahey, J.M. Cappiello Dziedzic, L. Oh, and E. Greenhalgh, 

are with the Chemistry Department at Wake Forest University in Winston-Salem, North Carolina and Ameritox, LLC, in Greensboro, North Carolina. 

Oneka T. Cummings, Erin C. Strickland, and Gregory L. McIntire 

is with the Chemistry Department at Wake Forest University. Direct correspondence to: 

Articles

A novel “dilute-and-shoot” LC–MS/MS method is described for the analysis of “bath salts” sold as “legal” highs, including mitragynine and nine synthetic cathinones, in urine.

Liquid Chromatography Tandem Mass Spectrometry Method for Novel Psychoactive Substances: Kratom and Synthetic Cathinones in Urine

Naltrexone (Depade, Re Via, and Trexan) is a potent narcotic antagonist structurally similar to oxymorphone and naloxone. It blocks the subjective effects of heroin and other opiates and is primarily used in the management of opioid dependence and alcohol dependence. While conjugated 6β-naltrexol is the major urinary metabolite in man, conjugated naltrexone and free 6β-naltrexol are also major urinary species. To assist with monitoring substance abuse patients who are prescribed naltrexone, a rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed to analyze for both naltrexone and 6β-naltrexol post-enzymatic hydrolysis. The validation of this method and some representative patient data are discussed in this article.

	Naltrexone is a pure narcotic antagonist structurally similar to oxymorphone and naloxone. It blocks the subjective effects of heroin and other opiates and is primarily used in the management of opioid and alcohol dependence (1). The drug is supplied as 25-mg or 50-mg tablets (under the brands Depade, ReVia, and Trexan) (2) and as an extended-release injectable formulation (Vivitrol) (3), which is administered monthly as an intramuscular injection (1). Naltrexone undergoes extensive first-pass metabolism, and up to 70% of a dose can be recovered in urine within 24 h (1,4). Conjugated 6β-naltrexol (naltrexol) is the major urinary metabolite in humans, and conjugated naltrexone and free naltrexol are other known major urinary species (5). Naltrexol has significant pharmacological activity with a half-life that is greater than three times its parent, naltrexone (6).

	Many of the published analytical methods used to detect naltrexone and its metabolites were reported in plasma, which is a more complex matrix and usually requires more sample processing and cleanup when compared to urine (5,7–9). In a high-throughput laboratory environment, the emphasis is on developing methods that are not only rapid but also sensitive and selective. In view of that, this work demonstrates a method that requires dilution with internal standard, hydrolysis, and centrifugation. The subsequent analysis of naltrexone demonstrates toxicologically acceptable limits of quantitation (LOQs) and limits of detection (LODs) and robust instrument performance that minimizes instrument utilization time when analyzing naltrexone in urine.

	The resulting method reported herein has a 1.7-min run time and a 2.7-min cycle time using a liquid chromatography–tandem mass spectrometry (LC–MS-MS) system. Limits of quantitation for both naltrexone and 6β-naltrexol were determined to be 5 ng/mL. The development and validation of this method are discussed in further detail in this article.

	Reference standards for naltrexone, naltrexone-D3, 6β-naltrexol, oxymorphone glucuronide, and oxymorphone were purchased from Cerilliant. All solvents, including methanol (Optima grade), acetonitrile (Optima grade), isopropanol (Optima grade), 0.2 M phosphate buffer (pH 7.5), and formic acid (88%) were purchased from VWR. Drug free urine was obtained from Utak Laboratories. IMCSzyme, a recombinant β-glucuronidase, was purchased from IMCS.

	The analytical method was developed using an Agilent 6460 LC–MS-MS system. After testing several ultrahigh-pressure liquid chromatography (UHPLC) columns, a 50 mm x 4.6 mm, 100-Å, 2.6-µm 

 Phenomenex Phenyl Hexyl column coupled with a Phenomenex guard cartridge (Phenyl) was used to validate this method. The column temperature was maintained at 50 °C during analysis.

	Patient urine specimens (50 µL) were diluted 10x with a mastermix consisting of 1.6 µg/mL of naltrexone-D3 and 2000 units of a recombinant β-glucuronidase from IMCS (IMCSzyme) in 0.02 M phosphate buffer, pH 7.5. Samples were incubated in an oven for 60 min at 60 °C for hydrolysis. Earlier studies using oxymorphone glucuronide (naltrexone glucuronide is not commercially available) demonstrated the time course of hydrolysis for these analytes required >60 min for complete hydrolysis. It was determined that a hydrolysis time of 60 min provided stable and consistent results with a control. A correction factor of 1.14-adjusting for approximately 88% hydrolysis efficiency-was applied to results to mimic complete hydrolysis. Samples were removed from the oven and centrifuged before analysis by LC–MS-MS.

	Reference standards were diluted to appropriate calibrator level concentrations (5, 10, 50, 250, 1000, and 5000 ng/mL) in normal human urine and were then diluted an additional 10x as detailed above. The standards were meant to mimic the already 10x diluted patient samples. An oxymorphone glucuronide hydrolysis control was used to ensure the enzyme–mastermix was acceptable for every batch in addition to a positive and negative control.

	This method was validated for naltrexone and naltrexol as per an internal standard operating procedure (SOP) based upon College of American Pathologist requirements (10) and applied to authentic positive urine samples to evaluate efficacy for adherence drug testing. For a standard at any concentration level to be considered passing, during the validation of this method, quantitation must be within ±25% of the expected value and ion ratios and chromatographic requirements must pass for all analytes assessed. Quantitation values are based on a passing calibration curve (no points excluded and 

 value greater than or equal to 0.99), which must be freshly prepared and analyzed on the same day that the particular validation study was conducted. Furthermore, the coefficient of variation (CV) between sets of replicates must be ≤15%. Experiments performed during the method validation process are explained in greater detail below.

	The lower and upper concentration limits at which the method could accurately identify and quantify analytes were evaluated. To establish reliable LODs and LOQs, four out of five replicates must pass for the necessary criteria when compared to a passing calibration curve (detailed above). The LOD and LOQ both require the analyte signal to demonstrate reproducible ion ratio stability, while the LOQ requires the analyte signal to demonstrate reproducible quantitation.

is the reproducible regression or fit of the calibration curve compared to expected concentration values. Four of the five replicates of each linearity point-usually comprising the calibrators and a minimum of five additional points within the analytical range-must pass all validation criteria.

 is the highest quantitative level of analytes present that do not produce a concentration level above the lower limit of quantitation in a subsequent blank injection. Carryover is investigated to ensure false positives are alleviated in a production environment. This experiment requires four of five blank injections following each of the five replicates at the potential carryover limit concentration to quantitate lower than the established LOQ.

	The precision and accuracy test measures the capability of the method to yield reproducibly accurate results over a period of multiple days at concentrations spanning the concentration range of interest. A total of 10 replicates of three concentrations (omitting calibration points) within the analytical range of interest are chosen and run on three separate days. Out of these 10 injections, nine must pass within 25% of the target concentration.

	The ability of the method to be unaffected by the presence of other medications or compounds is the 

. Interference would be considered confirmed if a false signal met or exceeded LOQ requirements (that is, was higher than the LOQ and had a passing ion ratio) in greater than one of the five replicates that must be run for each individual test.

 are the suppression or enhancement of analyte signals of interest because of the presence of matrix-in this case urine. The effect of matrix on the method was evaluated by running the quality control (QC) concentration of 750 ng/mL both in matrix and in the LC starting conditions (98% 5 mM ammonium formate in 10% methanol: 2% methanol).

	This test is used to confirm whether a new method accurately quantitates actual patient samples when compared to the currently in-use and accepted method for these analytes (10). The previously validated method used a Waters Acquity TQD system and had been in regular daily use for more than a year. To make this comparison, all recent patient positive urine samples for the analytes in this method were prepared using the new procedure and compared to the previous method.

	Daily calibration curves (5, 10, 50, 250, 1000, and 5000 ng/mL) routinely demonstrated 100 ± 25% accuracy for each point and excellent goodness of fit (

 > 0.99). Calibration curves for oxymorphone only included 250, 1000, and 5000 ng/mL as the hydrolysis control was set to liberate 1577.8 ng/mL. Consistent peak shapes and ion ratios were achieved for naltrexone, naltrexol, and oxymorphone. A typical chromatogram is shown in Figure 1 for the 100 ng/mL calibrator demonstrating the separation achieved for this 1.7-min method; note that naltrexol, the latest eluted, is retained well before 1 min of the gradient. The longer run time (1.7 min) and cycle time (2.7 min) are necessary for column equilibration and needle washing–carryover reduction, respectively. Details about the LC gradient and MS ion fragmentation transitions used are described in Tables I and II, respectively. Compiled results for all conducted method validation experiments are shown in Table III.

					Figure 1: Example chromatogram of a 100-ng/mL sample injection (internal standard excluded).

	The LOD and LOQ for naltrexone and naltrexol were determined to be 5 ng/mL and 100 ng/mL for oxymorphone. No appreciable carryover was observed following the highest curve point of 5000 ng/mL (see Table III). The precision and accuracy was evaluated by assessing 10 replicates of standards at 75, 750, and 2500 ng/mL on three separate days-usually there is at least one skipped day between precision and accuracy experiments. The precision and accuracy results shown in Table III demonstrate the robustness of this method for all three analytes at the three concentration levels tested.

	All analytes experienced suppressed signal response when dissolved in normal human urine compared to a neat preparation in mobile phase initial conditions (98% 5 mM ammonium formate in 10% methanol: 2% methanol). Different methods can be used to calculate the effect of the matrix on the signal response. To calculate the percentages presented in Table III the relationship between the absolute responses is used since it is believed to present a more thorough picture than comparison of relative responses or calculated concentrations.

	The interference study was conducted and there were no identified interferences for any of the analytes in this method. Interference analytes included structurally similar compounds such as naloxone, 6α-naloxol, codeine, oxycodone, noroxycodone, hydromorphone, hydrocodone, and norhydrocodone as well as panels of common drugs of abuse such as opiates, opioids, and benzodiazepines.

	In addition to the validation, the patient samples tested showed 100% qualitative correlation between the original method and the newly validated method. Results from patient samples submitted who tested positive using this method and had known prescriptions for naltrexone (tablets or injectables) are listed in Table IV. Of the 7158 patient samples analyzed over a one-month period in a production setting using this method, 125 had known prescriptions and 90 tested positive while also passing all the specimen validity criteria (11,12). The summary of this sample set (positive patients with a known prescription) is presented in Table IV.

	Analysis of naltrexone dosages show a range of 15 ng/mL to 42,017 ng/mL with a mean of 4506 ng/mL for daily tablets (50 mg taken between once and thrice daily) and a narrower range of 18 ng/mL to 18,389 ng/mL for the injectable Vivitrol formulation with a mean of 1381 ng/mL. It is difficult to assess a mean for these data because of the large discrepancies between measured concentration values for patients with both dosage forms. The median is a more meaningful measure for these data and is higher for patients taking the injectable form of naltrexone when compared to those taking the daily tablets.

	This work demonstrates that the analysis of naltrexone and its major metabolite, 6β-naltrexol, can be adequately assessed in a rapid LC–MS-MS quantitative method following hydrolysis. Key features of the method include hydrolysis using a recombinant enzyme and a cycle time of 2.7 min while achieving the high level of selectivity and specificity required for naltrexone and naltrexol. The results from this method compare well with the same samples run with an LC–MS-MS method on a different system. This newly developed method uses different transitions and a comparable column and reduces the cycle time by approximately 1 min.

Disposition of Toxic Drugs and Chemicals in Man

, 10th Edition (Biomedical Publications, Seal Beach, California, 2014), pp. 1419–1420.

		Depade (package insert), Hazelwood, Missouri, Mallinckrodt Pharmaceuticals Inc. (2014).

		Vivitrol (package insert), Waltham, Massachusetts, Alkermes Inc. (2010).

		K. Verebay, M.J. Kogan, A. de Pace, and S.J. Mule, 

		W. Huang, D.E. Moody, R.L. Foltz, and S.L. Walsh, 

		E.J. Cone, C.W. Gorodetzky, and S.Y. Yeh, 

		A.F. Davidson, T.A. Emm, and H.J. Pieniaszek, 

		Substance Abuse and Mental Health Services Administration Department of Health and Human Services, “Mandatory Guidelines for Federal Workplace Drug Testing Programs,” Federal Register 

 are with Ameritox, Ltd., in Greensboro, North Carolina. Direct correspondence to: 

Naltrexone (Depade, Re Via, Trexan) is a potent narcotic antagonist structurally similar to oxymorphone and naloxone. It blocks the subjective effects of heroin and other opiates and is primarily used in the management of opioid dependence and alcohol dependence. While conjugated 6β-naltrexol is the major urinary metabolite in man, conjugated naltrexone and free 6β-naltrexol are also major urinary species. To assist with monitoring substance abuse patients who are prescribed naltrexone, a rapid, selective, and sensitive LC–MS-MS method was developed to analyze for both naltrexone and 6β-naltrexol post-enzymatic hydrolysis. The validation of this method and some representative patient data are discussed in this report.

Oneka T. Cummings

Articles

Liquid Chromatography Tandem Mass Spectrometry Method for Novel Psychoactive Substances: Kratom and Synthetic Cathinones in Urine

A Rapid, Selective, and Sensitive Method for the Analysis of Naltrexone and 6β-Naltrexol in Urine