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Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 4819599, 9 pages https://doi.org/10.1155/2022/4819599 Research Article A Human Oral Fluid Assay for D- and L- Isomer Detection of Amphetamine and Methamphetamine Using Liquid- Liquid Extraction 1 2 1,3 4 Brian Robbins , Rob E. Carpenter , Mary Long , and Jacob Perry Department of Research, Advanta Genetics, 10935 CR 159 Tyler, Texas 75703, USA University of Texas at Tyler, 3900 University Boulevard, Tyler, Texas 75799, USA University of Alabama at Birmingham, 1720 University Blvd, Birmingham, AL 35294, USA University of Miami Miller School of Medicine, 1600 NW 10th Ave, Miami, FL 33136, USA Correspondence should be addressed to Rob E. Carpenter; firstname.lastname@example.org Received 6 October 2022; Revised 3 November 2022; Accepted 12 November 2022; Published 2 December 2022 Academic Editor: Nu ´ ria Fontanals Copyright © 2022 Brian Robbins et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medical providers are increasingly confronted with clinical decision-making that involves (meth)amphetamines. And clinical laboratories need a sensitive, efcient assay for routine assessment of D- and L-isomers to determine the probable source of these potentially illicit analytes. Tis paper presents a validated method of D- and L-isomer detection in human oral fuid from an extract used for determination of a large oral fuid assay (63 analytes) on an older AB SCIEX 4000 instrument. Taken from the positive extract, D- and L-analytes were added. Te method for extraction included addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. Te samples were suspended in 50% MeOH in water, diluted with mobile phase, with separation and detection accomplished using LC-MS/MS to determine analyte con- centration. Once samples were confrmed positive for (meth)amphetamine from the large oral fuid assay, they were further examined for the enantiomeric forms with 50μl aliquots of the standards and samples of interest combined with 450μl of D- and L-assay mobile phase, then analyzed using chiral column separation, and LC-MS/MS detection with standard curve spanning the range from 2.5 to 1000 ng/mL. Te result is a sensitive and accurate detection of D- and L-isomers of amphetamine and methamphetamine in human oral fuid performed on an older model mass spectrometer (AB SCIEX 4000). Te novelty of this assay is twofold (a) the 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples, and (b) its adoption characteristics as a refex test from a large ODT panel without the need to invest in newer or expensive LC-MS/MS instruments. Finally, this assay also has potential to add a valuable option to high-throughput laboratories seeking a D- and L- testing alternative to urine drug testing methods. Vicks VapoInhaler . Moreover, the D-form can be used 1. Introduction therapeutically as a treatment for overeating disorders, Amphetamines and methamphetamine are popular illicit narcolepsy, and attention defcit disorder, but produces the drugs of abuse for their stimulation of the central nervous sought-after habit-forming central nervous system (CNS) system. Tese central nervous system (CNS) stimulants exist efects that can be abused and long term neurotoxicity [2–4]. as two enantiomeric forms, dexter (D-) or laevus (L-), which Tus, medical providers are increasingly confronted with produce radically diferent efects on the human system. clinical decision-making that involves (meth)amphetami- Notwithstanding the potential use disorder from the nes—including illicit use. heighted dopamine response that D- (meth)amphetamine Routine assessment for noncompliance or nonmedical asserts [1, 2], the L-form is quite common and is an efective use of (meth)amphetamine is frequently accomplished vasoconstrictor used in the over-the-counter formulation of through urine drug testing (UDT) based on risk of drug 2 Journal of Analytical Methods in Chemistry methodologies may be more economic for this purpose, this misuse, abuse, and diversion. Although UDT is considered the common practice for detecting scheduled drug com- assay provides small resource-limited laboratories with older model AB SCIEX 4000 instruments to operationalize dis- pliance or illicit use, often medical providers are unable to procure a urine sample for various reasons. In this case, oral covery of enantiomeric forms of D- and L- (meth)am- fuid drug testing (ODT) can serve as an efective alternative phetamine without incurring additional equipment to UDT . ODT is increasingly emerging as an alternative expensive. biological matrix for detecting drugs and monitoring patient medication compliance [6–8]. Although the detection 2. Methods and Materials window for oral fuid is small compared to matrices like 2.1. Reagents and Standards. All analyte stock solutions at urine, hair, or sweet, in certain clinical situations—like 1 mg/mL concentrations and deuterated internal standards immediate detection of recent marijuana use—oral fuid at 100μg/mL were purchased from Cerilliant Corporation shows to be more benefcial over UDT [9, 10]. Te matrix (Round Rock, TX, USA). All organic solvents including allows for easy collection, but attention to recovery, stability, methanol, acetonitrile, formic acid (88%), dichloromethane, and dilutions issues of some collection devices should be 2 propanol, and ethyl acetate were obtained from Fisher given consideration for pharmacokinetic studies [10, 11]. Scientifc (Pittsburgh, PA, USA). Because experiments have Although ODT opioid assays that use dilute-and-shoot demonstrated considerable variation in recovery of (meth) methods with little sample manipulation have been devel- amphetamine from various oral fuid collections devices oped and validated on AB SCIEX 4500 instruments with , the Quantisal oral fuid collection device was used excellent calibration ranges (2.5–1,000 ng/mL)  robust based upon its exhibited recovery of (meth)amphetamine ODT assays that also quantify the D- and L-isomers of shown to exceeded 93%, and the device has seen good re- (meth)amphetamine are less common. Notably, there are covery in liquid-liquid extraction techniques [15, 16]. Te several methods available for determining D- and L- (meth) Quantisal oral fuid collection device and extraction bufer amphetamine including immunoassays which have been were obtained from Immunalysis Corporation (Pomona, designed to react with one or the other but often have CA, USA). problems with cross-reactivity. Gas chromatography–mass spectrometry (GC-MS) can also be used but often requires an extra step requiring the synthesis of derivatives resulting 2.2. Mobile Phase and Extraction Solutions. A D- and L- in potential purity issues and errors in concentration esti- mobile phase (MPDL) solution was created by adding mation. Other methodologies including some liquid chro- ∼993.2 mL of methanol to a 1 L bottle. Ten using a 1 mL matography with tandem mass spectrometry (LC-MS/MS) pipettor, 5 mL of type I water, 1.5 mL of acetic acid, and methods also require purifcation steps with solid phase 0.3 mL of ammonium hydroxide were added. Tis solution extraction cartridges involving additional equipment and can be kept at room temperature for up to 1 year. Extraction the cartridges themselves adding extra cost per sample. solution 1 (ES1) was created with 50% dichloromethane and Accordingly, there is a need for developing an efcient 50% 2-propanol by using a graduated cylinder under a fume method to detect enantiomeric forms of D- and L- (meth) hood. Equal volumes of dichloromethane and 2-propanol amphetamine, especially in small resource-limited labora- were added to a clean reagent bottle which was capped and tories without the capacity to invest in newer LC-MS/MS mixed well. Extraction solution 2 (ES2) was created with instruments. 50% dichloromethane and 50% ethyl acetate by using a In this paper, we ofer a novel approach for a fast, ac- graduated cylinder under a fume hood. Equal volumes of curate, and applicable method to quantify the D- and L- dichloromethane and ethyl acetate were added to a clean isomers of (meth)amphetamine in human oral fuid speci- reagent bottle which was capped and mixed well. mens using liquid-liquid extraction and LC-MS/MS with an older model AB SCIEX 4000 instrument as an extension of larger ODT assay. A preprint version of this assay paper has 2.3. Standard Preparation. An 8000 ng/mL stock solution been published . Each patient sample was initially an- was made by combining analyte stock controls and diluting alyzed for 63 targeted analytes using LC-MS/MS with the it with MPA. In contrast, D- and L-amphetamine and same extracts injected a second time using a delta-9 tetra- methamphetamine were added in an amount to make a hydrocannabinol (THC)-specifc (ES) negative assay to 4000 ng/mL stock of each isomer so that when combined capture the THC. Ten, for samples that exhibited a positive they would produce an 8000 ng/mL solution of total am- confrmation result for (meth)amphetamine, an additional phetamine and methamphetamine. Tis means that the sample was taken from the previously extracted specimen. range of the D-and L standard curve (SC) is from 2.5 to Tis sample was then analyzed with a newly developed assay 1000 ng/mL (half the concentration). Te resulting stock designed specifcally to assess the D- and L-isomer status to standard was diluted with mobile phase A (MPA) to produce defne nonillicit versus illicit etiology. Te assay develop- the SC. Concentrations were 8000 (undiluted), 4000, 2000, ment and validation ofered here is for the beneft of high- 1000 400, 200, 100, 40, 20, 10, 4, and 2 ng/mL. Tese so- throughput laboratories seeking novel solutions for a lutions were stored at the concentrations above. Tey un- quantitative D- and L-isomer test from oral fuid with fast derwent a dilution during the assay (1 part standard to 3 and accurate chemical analysis using less expensive older parts mobile phase and THC standard) to achieve the model AB SCIEX instruments. Although other concentration desired in sample analysis with oral fuid Journal of Analytical Methods in Chemistry 3 (saliva). Te standards and quality control (QC) were diluted analysis by LC-MS/MS involved transferring 50μL of the (0.5 mL) with 1.5 mL of extraction bufer. Tis approximates already extracted standards, QC, and any samples of interest the condition seen with saliva after collection with the to a new plate. Ten 450 μL of MPDL was added to each well Quantisal oral fuid sample collection device. Te fnal and mixed with a multichannel pipette, the plate was cov- concentration in the 0.5 mL sample SC included the fol- ered with a plate mat and analyzed for the D- and L-isomers lowing points: 2000, 1000, 500, 250, 100, 50, 25, 10, 5, 2.5, 1, of amphetamine and methamphetamine using the listed and 0.5 ng/mL. chiral column. Te LC-MS/MS conditions and separation Te assay QCs were made similarly; frst making a parameters are presented in Table 1. 7200 ng/mL spiking solution in MPA then diluting to 3200, 2400, 300, 60, 12, and 2 ng/mL. Te D- and L-amphetamine 3. Method Validation Procedures and methamphetamine QCs were made at half concentra- Te assay was developed as an extension of a larger 63 tions. Final concentrations of each QC were 1800, 800, 600, analyte assay whereby samples that were confrmed positive 75, 15, 3, and 0.5 ng/mL, after the 1 : 4 dilution with MPA for (meth)amphetamine could be refex tested to identify and THC QC same as the SC points noted above. nonillicit versus illicit etiology. Te assay was initially op- Te internal standard working solution (ISWS) for the timized for extraction of all the required analytes in the large oral fuid assay and the D- and L-assay was made by larger panel. Samples were spiked with a combination of all flling a 100 mL graduated cylinder to the 50 mL mark with the drugs of study and multiple solvent systems and single 10% methanol in water and adding 250μL of each of the and two step processes were evaluated including the com- internal standards listed above. Te volume was brought to binations fnally selected; these were dichloromethane plus 100 mL with additional 10% methanol producing a con- isopropyl alcohol for solution 1 and dichloromethane plus centration of 250 ng/mL. ethyl acetate for solution 2 as these appeared to be the most broad-spectrum solvents for extraction of the diferent 2.4. Instrumentation. Te liquid chromatography compo- analytes tested—including methamphetamine and its me- nents of the LC-MS/MS system consisted of a model CBM- tabolite amphetamine, and analytes with diferent chemical 20A controller, 2 model Prominence LC-20AD pumps, a solubility characteristics such as meprobamate. Moreover, model DGU-20A5 degasser, and a model SIL-20AC auto- the D- and L-isomer component of this assay was adapted sampler all obtained from (Shimadzu, Columbia MD, USA, from a dilute and shoot method for analysis of urine samples based in Kyoto, Japan). Te mass spectrometer used was a and was modifed to the back end of the oral fuid assay after SCIEX API 4000 and the acquisition software was Analyst, v extraction. Importantly, it was found during development 1.5.2, build 5704 (Framingham, MA, USA). Nitrogen was that the elimination of phentermine was achieved by both obtained using a Peak ABN2ZA gas generator (Peak Sci- chromatographic separation and elimination by careful entifc, Billerica, MA, USA). Reagents were weighed on a selection of the secondary transition. Mettler Toledo MX5 analytical micro balance (Fisher Sci- entifc, Pittsburgh, PA, USA). Samples were dried on a 3.1.MatrixLot-to-LotComparison. Individual lots of human TurboVap LV (Uppsala, Sweden). Samples were vortexed matrix (saliva) difer according to a person’s overall health on a Fisherbrand 120 multitube vortex. Te analytical col- and hydration status . A single lot of oral fuid is not umn was an Astec CHIROBIOTIC V2 5.0μm enough to demonstrate the ruggedness of the assay system (2.1 mm × 25 cm column) Catalog # 15020AST SUPLECO , when such variability in the matrix exists . Due to this, (Bellefonte, PA, USA). and in accordance with the current College of American Pathologists (CAP) standards, a minimum of 10 lots of 2.5. Analyte Optimization. Individual analytes and internal human matrix were collected from drug-free donors. Tese standards were optimized by using T-infusion with 50% B oral fuid samples were spiked at a low-level concentration mobile phase and tuning for declustering potential (DP), with each analyte. Tese samples were prepared, extracted, entrance potential (EP), collision energy (CE), and exit and run as described above. Te responses were calculated potential (CXP) at a fow rate of 0.7 mL/min. Te two most and the analyte to internal standard (IS) ratio and %CV is abundant fragments were selected for monitoring using shown in Table 2. multiple reaction monitoring (MRM). 3.2. Analytical Measurement Range. Te analytical mea- surement range (AMR) of the assay refers to the concen- 2.6. Sample Preparation and Procedures. Samples were collected using the Quantisal oral fuid collection device. tration range that the assay is validated within and is Te manufacturers collections instructions were followed determined by running a series of calibration curve stan- (https://immunalysis.com/products/oral-fuid/quantisal/). dards covering a concentration range that encompass the Te samples, standards, and QC were extracted using two concentration of analyte expected to fnd in patient samples liquid-liquid extractions with 1 : 1 DCM : IPA and 1 : 1 . Te limits of the AMR were bounded by the lower limit DCM : EtAc. Tey were combined, dried, reconstituted with of quantitation (LLOQ) and the upper limit of quantitation 50% MeOH water, and combined with mobile phase for (ULOQ). Te dynamic range may be described by a linear or separation of the analytes. Sample preparation for D- and L- quadratic ft [19, 20]. Calibration curves were created using a 4 Journal of Analytical Methods in Chemistry Table 1: LC-MS/MS conditions for the three assays on a single oral fuid sample. D- and L- Scan type MRM Ion source Turbo spray Probe position X � 5.00, Y � 5.2 Polarity Positive Run duration 11 min Settling time (msec) 0 Pause time (msec) 7.007 msec Curtain gas 35 CAD gas 4 ISV (V) 5000 Temperature ( C) 500 Ion source gas 1 (GS 1) 50 Ion source gas 2 (GS 2) 50 Q1/Q3 resolution: unit/unit CEM (V) 2600 Inlet settings Analytical column Supelco Astek Chirobiotic V 250 × 2.1 mm, 5μm Guard cartridge None Sample temperature 15± 5.0 C Column temperature 30.0± 5.0 C Mobile phase A Water : acetic acid : ammonium hydroxide : Methanol 5 : 1 : 0.3 : 993.5 Mobile phase B N/A Needle rinse Water : acetic acid : ammonium hydroxide : Methanol 5 : 1 : 0.3 : 993.5 Flow rate 0.3 mL/min Injection volume 10μL Run time 11 min Note. collision gas (CAD), ion source voltage (ISV), channel electron multiplier (CEM). Table 2: Matrix efects 10 diferent lots of oral fuid were fortifed detection. Te sensitivity of the assay system was tested by with QC material to a concentration of 7.5 ng/mL and the %CV injecting 6 replicates of the LLOQ over 3 days and evaluating determined of the analyte/IS area ratio. the resulting analytical determinations. Standard acceptance criteria of ±20% of nominal concentration apply. Matrix comparison Drug/Metabolite %CV analyte/IS ratio Amphetamine 3.39 ∗ 3.4. Intraday Precision and Accuracy. Intraday precision Methamphetamine 4.60 and accuracy were determined using six replicates of each D-amphetamine 1.59 of three QC sample determinations and LLOQ from L-amphetamine 2.35 D-methamphetamine 7.54 across at least three validation runs. Concentrations of the L-methamphetamine 3.09 QC samples ranged across the curve, with the low QC set Indicates combined D- and L-. at approximately 3 times the LLOQ or less, the mid QC near the mid-range of the linear range of the curve, and the high QC set at 80–90% of the ULOQ. Percent accuracy minimum of six nonzero calibration points. To be accepted and precision were determined for each individual as the AMR, all points describing the calibration curve must measurement. To be accepted, the precision and accuracy pass within ±20% of the nominal concentration . Fur- for the replicate determinations must be≤ 20% at each level. thermore, the correlation coefcient (R ) for the calibration curve must be≥ 0.99, or R should be≥ 0.98 to be acceptable [21, 22]. 3.5.InterdayPrecisionandAccuracy. Interday precision and accuracy were determined using all replicates of each of three quality control (QC low, QC mid, and QC high) and 3.3. Sensitivity. Te sensitivity of the assay system refers to the ability to reliably produce a signal throughout the entire LLOQ sample determinations from the analytical runs performed on 3 separate days. Concentrations of the QC calibration range, but specifcally at the low-end of the calibration curve (the lower limit of quantitation, LLOQ) samples ranged across the curve, with the low QC set around . In hyphenated mass spectrometry assays, a signal that 3 times the LLOQ, the mid QC near the middle of the linear range, and the high QC set at 80–90% of the ULOQ. To be produces a signal to noise ratio (S/N) of ≥10 is considered valid for the LLOQ of an assay system . Further, an S/N accepted, the precision and accuracy for the replicate de- terminations must be≤ 20% at each level. ratio of ≥5 is considered clear enough for the limit of Journal of Analytical Methods in Chemistry 5 3.6.ExogenousInterferingSubstances. Drugs that are known 4.2. Accuracy and Precision, LLOQ. Six replicates of each or suspected of interfering with similar bioanalytical systems validation level were run on at least 3 days. Te theoretical should be evaluated to ensure that they do not suppress concentrations were 1, 5, or 25 for LLOQ on the combined ionization or cause false-positive results for a given analyte concentrations, 3, 15, or 75 for QC low, 600 ng/mL for QC [25, 26]. Te following medications were evaluated: over- mid, and 800 or 1800 for the QC high values. Te D- and L- the-counter mix, acetaminophen, ibuprofen, pseudoephe- assay individually had an LLOQ of 2.5 ng/mL with a QC low drine, cafeine, and naproxen. Te following individual of 7.5 ng/mL, a QC mid of 300 ng/mL, and a high QC of analytes were also tested: salicylic acid, phenylephrine, 900 ng/mL. Tables 4–6 indicate mean, interassay, and phentermine, diphenhydramine, and dextromethorphan. A intraassay statistic variability were all below 20%. high concentration of the possible interfering drug (typically 2,000 ng/mL or greater) was spiked into a low QC sample 4.3. Partial Volumes Accuracy and Precision. An MPA sur- (15–75 ng/mL low QC). Acceptance criteria for a substance rogate sample was prepared at 4000 ng/mL. To determine the to be deemed as noninterfering is that the quantitated value concentration of this sample, a dilution must be made so the for the low QC should be within ±20% of the nominal value fnal concentration would be less than 2000 ng/mL to get it in . Furthermore, the spiked substance should not cause a the measurement range of the assay. Tree replicates of four false-positive or a false-negative result. dilutions were made and tested: (1) 1 : 5 target 400 ng/mL; (2) 1 : 10 with a target of 200 ng/mL; (3) 1 : 20 with a target of 100 ng/ mL; and (4) 1 : 50 with a target of 40 ng/mL. Te results shown 3.7. Partial Volumes and Dilutions. A spiked solution was in Table 7 indicate that all analytes can be diluted at all levels. created at a concentration above the ULOQ, in this case, 4000 ng/mL. Te sample was run at discrete dilutions of 1 : 5, 1 : 10, 1 : 20, and 1 : 50. Concentration determinations for all 4.4. Room Temperature, Refrigerator, and Freezer Stability. Samples with concentrations of 75, 800, or 1800 ng/mL were dilutions should be within ±20% of the nominal value following correction for the dilution factor [27, 28]. More prepared in triplicate. One set was kept at room temperature overnight (RT), a second set was kept in the refrigerator recent literature suggests that the signal to noise ratio of both the quantifcation trace and the qualifying ion trace be 3–10 overnight (RF), and a third set was kept in the freezer . On occasion, an analyte will not have a quantifying ion overnight (FZ). Tese validation samples were then run and compared to a triplicate preparation of QC samples that had that passes this criterion while still permitting the quanti- fcation trace to remain in a meaningful range. Tese in- been analyzed as normal. All results show less than 20% deviation from expected (Table 8). stances should be documented in the laboratory standard operating procedure or validation report. 4.5. Freeze-Taw (FT) Stability. Validation samples with concentrations of 75, 800, or 1800 ng/mL were frozen at 3.8. Carryover. Carryover is the presence of an analyte in a −20 C and thawed in sequence with samples taken after each blank injection following a positive injection, resulting in a freeze-thaw cycle for a maximum of three cycles. Tese false-positive sample . Te injection needle should be validation samples were analyzed in triplicate and compared washed in-between samples with a needle wash solution that is to a triplicate preparation of validation samples that had not intended to remove contamination from the surface of the been subjected to this freeze-thaw cycle. Te experimental needle. Te efciency of this process is monitored during results showed all meeting acceptance criteria. validation by assessing carryover in the following manner. Samples are injected in the following sequence: high QC, wash, 4.6. Extracted Sample Stability. A stability experiment was high QC, wash, high QC, wash. Peak areas are integrated for performed where samples were stored in the instrument (3 both the analyte and internal standard. Peak area in the wash day) or refrigerator (7 day) and reinjected after 3 and 7 days. solutions should be 0.1% or less of that found in the high QC All samples were within 20% of the initial results. standard. In addition, the mean of the peak area in the three wash solutions following the high QC replicates should be less than 20% of the LLOQ being used for the assay . 4.7. Stability in Matrix. A series of triplicate samples were analyzed over 7 days for stability at room temperature, 4 C 4. Results and −20 C. Te results indicated that all analytes were stable for at least 7 days refrigerated and frozen. Te analytes were 4.1. Interday Average back Calculated Calibration Standards. stable at room temperature for 24 hours. Each validation run contained calibration standards with theoretical concentrations of 1, 2.5, 5, 10, 25, 50, 100, 500, 1000, and 2000 ng/mL of each of the analytes with an additional 4.8. Matrix Recovery/Matrix Efects. Table 9 indicates the negative run at 0.5 ng/mL. Table 3 shows the range of standard efect of 10 diferent matrix lots tested by using a series of curves of the combined amphetamine and the individual D- 7.5 ng/mL samples prepared in water, MPA, and 10 diferent andL-analytes and the correlation information.D- andL-curve matrices. Te results were acceptable with less than 20% CV concentrations were half the above concentration ranging from across oral fuid, water, and MPA meeting acceptance cri- 0.25 (neg) to 1000 ng/mL. Mean R values were all at least 0.99 teria. Tis is likely due to dilution in 1.5 mL Quantisal indicating good ft to the data. extraction bufer before extraction. 6 Journal of Analytical Methods in Chemistry Table 3: Statistical analysis for each analyte standard curve over three assays. Drug/Metabolite Curve range (ng/mL) Mean R RSD Mean slope SD slope N Fit Amphetamine 5–1000 0.9990 0.0006 0.0594 0.0037 3.0000 Quadratic Methamphetamine 5–2000 0.9999 0.0001 0.0104 0.0004 3.0000 Quadratic D-amphetamine 2.5–1000 0.9994 0.0003 0.0104 0.0010 3.0000 Quadratic L-amphetamine 2.5–1000 0.9993 0.0006 0.0105 0.0010 3.0000 Quadratic D-methamphetamine 2.5–1000 0.9995 0.0008 0.0218 0.0021 3.0000 Quadratic L-methamphetamine 2.5–1000 0.9985 0.0021 0.0246 0.0031 3.0000 Quadratic Indicates combined D- and L-. Table 4: Interassay mean and standard deviation (SD) of validation samples. LLOQ (ng/mL) LQC (ng/mL) MQC (ng/mL) HQC (ng/mL) Drug/Metabolite Mean± SD Mean± SD Mean± SD Mean± SD Amphetamine 5.4± 0.5 16.7± 0.7 581.5± 24.6 802.4± 64.4 Methamphetamine 5.1± 0.4 15.1± 0.5 585.6± 25.9 1837± 85.7 D-amphetamine 2.8± 0.1 8.3± 0.4 295.5± 14.8 940.3± 33.4 L-amphetamine 2.8± 0.1 8.4± 0.3 294.5± 6.1 874.7± 25.7 D-methamphetamine 2.6± 0.1 7.6± 0.3 300.6± 12.1 999.5± 48.9 L-methamphetamine 2.6± 0.1 7.7± 0.4 301.6± 16.1 1013.9± 51.4 Indicates combined D- and L-. Table 5: Interassay precision and accuracy: precision and accuracy over 3 days with replicates of 6 for a total of 18 samples. LLOQ LQC MQC HQC Drug/Metabolite %CV %E %CV %E %CV %E %CV %E Amphetamine 9.25 7.37 4.31 11.13 4.22 −3.09 8.02 0.30 Methamphetamine 8.52 2.81 3.34 0.69 4.42 −2.40 4.66 2.08 D-amphetamine 3.95 13.09 4.54 10.70 1.80 −1.52 3.55 4.47 L-amphetamine 4.12 10.00 3.04 11.52 2.07 −1.85 2.94 −2.81 D-methamphetamine 4.44 3.17 3.98 1.30 4.04 0.21 4.89 11.05 L-methamphetamine 5.54 3.21 4.98 2.33 5.32 0.52 5.07 12.66 Indicates combined D- and L-. Table 6: Intraassay precision and accuracy: precision and accuracy over 3 days with replicates of 6 for each day. LLOQ LQC MQC HQC Drug/Metabolite %CV %E %CV %E %CV %E %CV %E MIN MAX MIN MAX MIN MAX MIN MAX MIN MAX MIN MAX MIN MAX MIN MAX Amphetamine 2.28 10.19 −2.20 13.20 2.72 5.62 9.07 12.43 2.06 5.57 −5.07 −0.91 3.46 8.89 −5.00 6.59 Methamphetamine 4.84 9.89 −2.60 7.53 1.71 4.41 −0.02 1.66 1.71 2.92 −6.31 2.48 2.10 4.41 −2.22 6.50 D-amphetamine 1.38 3.51 8.44 15.79 1.14 1.40 4.38 15.69 0.69 1.73 −2.89 -0.02 1.46 2.99 0.75 7.90 L-amphetamine 2.60 4.47 6.63 13.33 1.11 1.79 9.38 15.71 1.14 1.57 −3.13 0.31 1.46 2.33 −5.16 0.02 D-methamphetamine 1.28 4.42 −1.56 5.73 1.67 2.66 −3.19 4.96 1.75 2.21 −4.28 4.24 1.67 2.74 7.59 17.89 L-methamphetamine 1.66 5.19 −2.83 7.65 2.59 3.56 −3.14 6.69 0.55 0.99 −4.74 7.49 2.17 5.87 8.68 17.91 Indicates combined D- and L-. Table 7: Dilution study: percent diference from expected with a 4000 (2000) ng/mL standard diluted as indicated. All analytes are based at a 1 : 10 dilution. Drug/Metabolite 1 : 5 dilution 1 : 10 dilution 1 : 20 dilution 1 : 50 dilution Amphetamine 1.03 4.04 9.45 12.00 Methamphetamine −8.17 −0.32 3.13 0.66 D-amphetamine 2.38 5.97 9.87 12.19 L-amphetamine 2.56 7.39 10.81 9.60 D-methamphetamine −0.51 −3.58 −1.77 −3.63 L-methamphetamine 3.89 1.00 5.46 3.87 Indicates combined D- and L-. High concentration was 2000 ng/ml for individual D- and L-isomers. Journal of Analytical Methods in Chemistry 7 Table 8: Stability testing. QC samples were tested for stability after 3 freeze-thaw cycles. Tey were also tested overnight at the indicated temperatures. A 3- and 7- day postextraction study were also performed at 2–8 C. F/T 3 cycles Overnight stability (%) Postpreparation stability Drug/Metabolite ° ° QC % Dif RT 4 C −20 C Init % dif nom % Dif init day 3 % Dif init day 7 QC 75 0.69 2.48 4.91 3.63 8.95 −1.33 0.61 Amphetamine QC 800 6.92 6.21 8.19 0.45 −0.69 0.16 −1.78 QC 75 −2.38 1.39 -0.23 0.72 −1.25 2.61 6.13 Methamphetamine QC 1800 1.12 2.62 1.95 4.85 −2.22 −4.26 −1.39 Indicates high QC of 800. Table 9: Matrix efects 10 diferent lots of oral fuid were fortifed with QC material to a concentration of 7.5 ng/mL and the %CV determined of the analyte/IS area ratio. Matrix comparison Drug/Metabolite %CV Analyte/IS ratio Amphetamine 3.39 Methamphetamine 4.60 D-amphetamine 1.59 L-amphetamine 2.35 D-methamphetamine 7.54 L-methamphetamine 3.09 Indicates combined D- and L-. Table 10: Concomitant medications: the indicated medications prepared in methanol were spiked into a QC 7.5 standard and measured. Te data indicates percent diference from a QC standard spiked with blank methanol at the same volume as the drug standards. % dif from MEOH spike Drug/Metabolite Dextromethorphan Phenylephrine Diphenhydramine Salicylic acid Phentermine OTC mix Amphetamine −0.92 −0.06 −0.84 1.89 −0.06 1.39 Methamphetamine 6.15 3.95 6.10 2.67 −2.29 −1.34 D-amphetamine −0.77 −1.64 −2.00 1.06 −0.94 −3.90 L-amphetamine −1.97 −0.41 2.07 1.30 3.21 −0.54 D-methamphetamine −6.26 5.53 −6.10 −3.81 7.45 −5.96 L-methamphetamine −2.10 −0.24 −0.08 0.71 7.73 −0.12 4.9. Selectivity. Multiple drugs that might have a potential simple and reliable means of sample collection coupled with for interfering with the assay analytes were run in the assay. a reduced chance of sample adulteration. Oral fuid also Samples of 500μL of 7.5 ng/mL QC were placed in a series of provides a viable alternative for measurement in patients tubes to be run in triplicate. To the frst set, 50 μL of MeOH that cannot provide an adequate urine sample volume such was added to act as the control. To the remaining tubes, as catheterized patients. Te drawbacks of the oral fuid assay 50μL of sample containing dextromethorphan, diphenhy- are that it has a shorter detection window and requires a dramine, phenylephrine, salicylic acid, or combo (includes more sensitive assay. Accordingly, this paper demonstrates a acetaminophen, cafeine, chlorpheniramine, ibuprofen, developed and validated cost-efective means of analysis naproxen, and pseudoephedrine). Tese solutions were using older, less sensitive instruments (API SCIEX 4000) by obtained from Cerilliant and were at a concentration of using a 2 step liquid-liquid extraction method and con- 1 mg/mL each except for the over-the-counter mix which centration of the samples with a nitrogen dry-down and a was 100μg/mL. Each solution was diluted to 20μg/mL in resuspension step. Te method of development of this D- methanol and this solution was used to spike samples as and L-assay was validated in accordance with the United indicated above. All samples met the accepted criteria and States Food and Drug Administration and the College of are diplayed in Table 10 and Table 11. American Pathologists guidelines  with an LLOQ of 2.5 ng/mL and ULOQ of 1000 ng/mL. Te most important aspect of this assay was its specifcity. It has the ability to 5. Discussion reliably and defnitively diferentiate between the isomeric forms of methamphetamine and its metabolite amphet- Te determination of prescription medications and illicit amine, as well as common decongestants and weight loss substances is needed for medical compliance . And medication. Phentermine in particular is a positional isomer human oral fuid is one of the most noninvasive and easily of methamphetamine that laboratories need to ensure does observed sample collection methods. It provides a relatively 8 Journal of Analytical Methods in Chemistry Table 11: Transitions and retention times for D-and L-amphetamine and methamphetamine. Drug RT (min) Q1 Q3 DP EP CE CXP D-amphetamine (S) 6.50 136 91.000 35 10 25 6 L-amphetamine (R) 7.21 136 91.101 35 10 25 6 D-amphetamine D-11 6.51 147.2 98.100 35 10 26 7 L-amphetamine D-11 7.22 147.2 98.101 35 10 26 7 D-methamphetamine (S) 7.91 150 119.100 10 10 25 9 L-methamphetamine R 8.45 150 119.101 10 10 25 9 D-methamphetamine D-11 7.91 161 97.100 85 10 26 7 L-methamphetamine D-11 8.46 161 97.101 85 10 26 7 Note. Retention times are updated using the internal standard each time that the data are analyzed. 6.3e4 1.6e5 5.6e4 1.4e5 2.7e6 2.0e6 8.0e4 3.0e4 5.0e5 0.0 0.0 0.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time (min) Time (min) Time (min) Amphetamine Methamphetamine Phentermine Figure 1: MRM chromatograms of amphetamine, methamphetamine, and phentermine. not interfere in (meth)amphetamine confrmation. As a Conflicts of Interest positional isomer, it shares a molecular weight and fragment Te authors declare that they have no conficts of interest to pattern nearly indiscernible from methamphetamine by report concerning this study; however, the authors are either many LC-MS/MS methods. 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Journal of Analytical Methods in Chemistry – Hindawi Publishing Corporation
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