Reproducibility and Quantification of Illicit Drugs Using Matrix-Assisted

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Analytical Chemistry

To be submitted to Analytical Chemistry:

Reproducibility and Quantification of Illicit Drugs using Matrix-Assisted Ionization (MAI) Mass Spectrometry Shubhashis Chakrabarty,†,‡ Jessica L. DeLeeuw,‡ Daniel W. Woodall,‡ Kevin Jooss,‡ Srinivas B. Narayan,§ and Sarah Trimpin*,†,‡,ǁ †

MSTM, LLC., Newark, DE

‡

Department of Chemistry, Wayne State University, Detroit, MI

§

Detroit Medical Center: Detroit Hospital (DMC), Detroit, MI

ǁ

Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI

*Correspondence to: S. Trimpin, Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, USA E-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract: Matrix-assisted ionization (MAI) mass spectrometry (MS) provides a simple and sensitive method for analysis of low and high mass compounds requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically-assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.


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Introduction Matrix-assisted ionization (MAI) 1-3 mass spectrometry (MS) is a powerful new approach that can be applied to a wide array of analytical problems. In MAI, analyte is incorporated

into

a

small

molecule

matrix

similar

to

matrix-assisted

laser

desorption/ionization (MALDI),4 but unlike MALDI, which requires laser ablation of the matrix:analyte mixture, MAI produces gas-phase analyte ions when the sample is exposed to the vacuum inherent with any mass spectrometer.5,6 MAI is an extremely simple ionization method applicable with atmospheric pressure ionization (API) mass spectrometer by exposure of the sample to sub-atmospheric pressure at the inlet aperture or intermediate pressure sources commonly used for MALDI by direct insertion of the sample into vacuum. Multiply charged ions are produced, similar to electrospray ionization (ESI),7 so that commonly available API-MS instruments are applicable.8 With the proper matrix, there is no need for heat, voltage, or nebulizing gases to produce gas-phase ions.1-3,8 The high sensitivity, low operating costs, speed of analysis, and ease of use make MAI an important addition to ionization methods used in MS and potentially useful in further extending MS to areas such as medical diagnostics, where ease of use and low-cost are important attributes. Extending MS to medical diagnostic applications has important societal benefits because of the potential for both targeted and non-targeted approaches to detection of diseases, 9 drug overdose and monitoring, 10 metabolic disorders, 11 and biomarker detection.12 Because of its sensitivity and speed of analysis, MALDI-MS has been used as a laboratory diagnostic tool13,14 but MALDI-MS instruments are expensive and have difficulty quantifying small molecules due to significant chemical background from the



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matrix.15,16 Often, qualitative information is not sufficient to make decisions and draw conclusions on the proper course of action in a clinical setting where accurate and rapid quantification is required for validation.17 Liquid chromatography (LC) hyphenated with MS is frequently the method of choice for quantitative measurements using MS,18,19 especially because of its effectiveness in quantification of low abundant molecules in biological matrices. However, LC/MS requires significant sample preparation, is subject to a chromatographic time delay, and requires a high degree of operator expertise if the methodology is used as a general diagnostic tool.20 Since the discovery of MAI more than forty matrix compounds that spontaneously produce analyte ions at room temperature and pressure have been discovered. 21 Similar to MALDI, some matrices are found to have different selectivity, allowing the matrix to be tailored to the problem. For example, the matrix compound 3nitrobenzonitrile (3-NBN) shows selectivity for compounds with basic functionality such as peptides and many drugs,3,22 but discriminates against compounds without basic functionality such as compounds that commonly make up the chemical background in MS. On the other hand, 1,2-dicyanobenzene (1,2-DCB) efficiently ionizes compounds without basic functionality such as lipids.21 MAI is applicable to a variety of commercial mass spectrometers123,8,21-25 eliminating the need of an ion source, and producing mass spectra from complex samples such as blood, urine, and tissue.2,26,27 In addition, MAI has recently been shown to be capable of high-throughput and automated analysis,26 requiring only a few seconds per sample. MAI is hypothesized to spontaneously produce gas-phase ions from a solid matrix by a sublimation driven triboluminescence process when the matrix:analyte sample is exposed to vacuum.2,3,21


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Reproducibility is an important parameter in a method’s acceptance in any field, and especially in medical diagnostics, and is traditionally assessed by relative standard deviation (RSD) with values less than 20%. 28 Previous high-throughput MAI studies dealing with clinically relevant samples initially reported relatively high standard deviation (up to 29%) in experiments without the use of internal standards.26 Here, MAI is used to assess and compare the RSD of the ion abundances of a mixture of illicit drugs over a range of inlet temperatures, different matrices, employing two different mass spectrometers and compared with ESI and MALDI. The utility of MAI-MS for direct quantitative analysis is also demonstrated for a mixture of drug standards spiked in blank human urine, as well as a real newborn patient urine sample containing the drug hydrocodone.

Experimental Materials An infant urine sample (OP1) that tested positive for opioids as well as a blank urine standard, were provided by the Detroit Medical Center (Detroit, MI). Morphine, morphine-D3, oxymorphone, oxymorphone-D3, codeine, codeine-D6, hydrocodone, hydrocodone-D6,

oxycodone,

oxycodone-D6,

(-)-∆9-tetrahydrocannabinol,

(-)-∆9-

tetrahydrocannabinol-D3, (±)-11-nor-9-carboxy-∆9-tetrahydrocannabinol and (±)-11-nor9-carboxy-∆9-tetrahydrocannabinol-D3 were purchased from Cerilliant (Round Rock, TX). Additional drugs, clozapine, clozapine-D8, buspirone, buspirone-D8, and matrices 3-NBN, 2-nitrobenzonitrile (2-NBN), methyl 2-methyl-3-nitrobenzoate and methyl 5nitro-2-furoate were purchased from Sigma Aldrich (St. Louis, MO). The MALDI matrix 5 ACS Paragon Plus Environment


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α-cyano-4-hydroxycinnamic acid (CHCA) was purchased from Acros Organics (Pittsburgh, PA). HPLC grade water, methanol and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). An eTrack XYZ stage and NSC-M3 motion controller were purchased from Newmark Systems, Inc. (Mission Viejo, CA).

Sample Preparation and Introduction Methods For MAI reproducibility studies, mixtures of 0.25 and 4 ppm morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone were prepared, with and without an internal standard, in 50% aqueous methanol, and blank human urine. The matrices 3NBN and methyl 5-nitro-2-furoate were prepared in acetonitrile at a concentration of 40 mg mL-1, while 2-NBN and methyl 2-methyl-3-nitrobenzoate were prepared in the same solvent at 100 mg mL-1. A 1 µL mixture of analyte and matrix (1:1, v:v) was introduced, after drying, to the inlet of the atmospheric pressure mass spectrometers via either a hand held pipette tip8,26 for the SYNAPT G2 or a gas chromatography (GC) injection needle adhered to programmable eTrack XYZ-stage for the LTQ Velos, adopted from the MSTM (Newark, DE) MAI sample introduction platform. From the same solution, 1 µL matrix:analyte was spotted onto a stainless steel MALDI plate for analysis using the SYNAPT G2 intermediate pressure source. For ESI reproducibility comparisons, the same 4 ppm mixture of the drug compounds, with and without internal standards, was diluted to 2 ppm in 50% aqueous methanol. The solution was introduced to the mass spectrometer by syringe infusion at a flow rate of 20 µL min-1. The MALDI experiments also used the 4 ppm drug mixture which was diluted 1:1 (v:v) with the matrix CHCA, prepared in 50% aqueous acetonitrile


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at a concentration of 16.7 mg mL-1 and 1 µL was spotted onto the MALDI plate. Each sample spot was completely consumed by ablating the entire area of each target spot with multiple laser shots using a software controlled sampling pattern (MassLynx v4.1). For MAI quantification studies of drug spiked urine samples, standards of (-)-Δ9tetrahydrocannabinol and ( ± )-11-nor-9-carboxy- Δ 9-tetrahydrocannabinol, morphine, codeine, oxymorphone and oxycodone along with equal concentrations of the respective deuterated internal standard were prepared by dilution in 50% aqueous methanol and spiked into samples of blank human urine; 10 ppm for cannabinoids and 5 ppm for opioids, respectively. An infant urine sample that tested positive for opioids using LC-MS19 was divided into 5 equal aliquots with 1 ppm of hydrocodone-D6 added, and spiked with increasing concentrations of hydrocodone (0, 1, 2, 3, and 4 ppm) to perform standard addition quantification. Each sample was mixed with the 3-NBN matrix solution (1:1, v:v), dried on a pipet tip, and introduced to the Z-Spray inlet aperture of the SYNAPT G2 mass spectrometer. Additional information regarding the mass spectrometers used and the data analysis can be found in the Supporting Information.

Results and Discussion Reproducibility Study of Illicit Drug Standards Using a 4 ppm mixture of six representative illicit drug standards (Scheme 1.I) with the 3-NBN MAI matrix (Scheme 1.II.A), RSD of the relative ion abundances was measured at different inlet temperatures using either a pipet tip or GC needle to introduce the matrix:analyte sample to the inlet of two commercially available mass spectrometers. Inlet temperatures studied were 30, 50, 80, 100, and 150 °C for the 7 ACS Paragon Plus Environment


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SYNAPT G2 and 50, 75, 100, 150, and 200 °C for the LTQ Velos. The relative ion abundance of each analyte was averaged over 15 data acquisitions at each of the five temperature settings for each instrument. On the Z-Spray source of the SYNAPT G2 the maximum ion abundance was observed for all drug compounds studied at 30 °C (Supporting Information Figure S1.A). As the source temperature was increased, the relative ion abundances of all analytes decreased, resulting in higher RSD values. The RSD values calculated with internal standards ranged from 2.4-9.9% at 30 °C (Supporting Information Figure S1.B). At this temperature, buspirone showed the lowest RSD value of 2.4%, followed by codeine, oxycodone, morphine, clozapine and finally oxymorphone at values of 3.6%, 4.8%, 5.6%, 6.6% and 9.9%, respectively (Supporting Information Table S1). At the maximum source temperature of 150 °C, the ion abundance and the reproducibility are the lowest. For experiments on the LTQ Velos, instead of introducing the sample to the inlet aperture using a pipette tip, a syringe needle was used to guide the sample into the inlet aperture (Supporting Information Scheme S1, Movie S1). This approach offers a more reproducible means of sample introduction without concern for melting a pipette tip when using high inlet tube temperature. Interestingly, using the LTQ Velos, the ion abundances of all drugs is the highest at an inlet tube temperature of 150 °C (Supporting Information Figure S2.A). The lowest RSD values were observed at 50 °C and 150 °C (Supporting Information Figure S2.B), but 150 °C was used because of the higher ion abundances (Supporting Information Figure S2.A). At 150 °C, clozapine showed the lowest RSD value of 3.1%, followed by oxycodone,


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oxymorphone, codeine, morphine and buspirone at values of 3.2%, 3.4%, 3.6%, 4.5% and 4.6%, respectively (Supporting Information Table S2). The lower RSD values observed with the LTQ Velos is likely related to the more consistent sample introduction method using the GC needle and a mechanically-assisted sample introduction stage. Similarly low RSD values were obtained using a lower concentration of the drug mixture on both mass spectrometers. The same respective introduction methods were used to analyze 15 replicates of the 0.25 ppm drug mixture. At 30 °C on the SYNAPT G2, the calculated RSD values were in the range of 2.4-24%. On the LTQ Velos, the range of RSD values was calculated to be 3.5-19% at 150 °C (Supporting Information Figure S3, Supporting Information Table S3). The higher ranges obtained are attributed to the lower concentration of each analyte in the drug mixture. For the subsequent reproducibility studies, an inlet temperature of 30 °C was used with the SYNAPT G2 and 150 °C for the LTQ Velos. The observed influence of temperature on ion abundance and reproducibility on the two mass spectrometers may be related to the different inlet designs; a skimmer cone inlet for the SYNAPT G2 and an ion transfer tube for the LTQ Velos. Differences in inlet geometries affect the pressure gradient from atmospheric pressure, where the samples are introduced, to the vacuum conditions inside the mass spectrometer. Pressure has been previously reported to play an important role in the MAI ionization process.2,3,8,21 The day-to-day reproducibility of MAI was determined by taking measurements over five consecutive days at the optimal temperature for the respective mass spectrometer using the matrix 3-NBN and analyte concentration of 2 ppm. The RSD values of MAI acquisitions were calculated to be in the range of 1.9-18% on the



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SYNAPT G2 (Figure 1.A). Due to the lower overall ionization efficiency of oxymorphone (Scheme 1.I.B) compared to the remainder of the mixture under the conditions used on the SYNAPT G2, on some days, this drug shows higher RSD values. The data acquired from the mixture, on days 1 to 5 ranged from 1.8-15%, 2.7-9.8%, 2.3-9.8%, 2.0-7.4%, and 1.9-18% (Supporting Information Table S4), respectively. On the LTQ Velos, RSD values of the same mixture obtained over five days using MAI ranged from 3.5-9.3% (Figure 1.B). The fluctuation in RSD between days 1 to 5 were 4.2-7.2%, 3.7-8.7%, 3.8-8.9%, 3.5-6.9%, and 4.1-9.3% (Supporting Information Table S5), respectively. The ion suppression of oxymorphone observed on the SYNAPT G2 was not observed to the same extent on the LTQ Velos. The RSD ranges of day-to-day studies are similar to data obtained in a single day on both mass spectrometers. This demonstrates the reproducibility of this novel ionization method using internal standards on two different mass spectrometers with inherently different source designs. RSD values for the drug mixture were also calculated using the MAI matrices3,21 2-NBN, methyl 2-methyl-3-nitrobenzoate, and methyl 5-nitro-2-furoate (Scheme 1.II). The matrix 2-NBN produced RSD values ranging between 3.1-18%, methyl 2-methyl-3nitrobenzoate between 2.8-30%, and methyl 5-nitro-2-furoate between 5.4-14% on the SYNAPT G2, in comparison to 2.4-9.9% for 3-NBN (Figure 2.A, Supporting Information Table S6). Methyl 2-methyl-3-nitrobenzoate was not ideal for consistently ensuring high ionization efficiency of oxymorphone at 30 °C. MAI-MS on the LTQ Velos gave RSD values for 2-NBN that ranged between 3.9-12%, methyl 2-methyl-3nitrobenzoate between 4.1-17%, and methyl 5-nitro-2-furoate between 8.8-32% as


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compared to 3-NBN of 3.1-4.6% (Figure 2.B, Supporting Information Table S7). Methyl 5-nitro-2-furoate failed to effectively ionize morphine at 150 °C. Despite these exceptions, the additional matrices can be considered for their success in reproducible ionization of drug mixtures using MAI and temperature conditions optimized for 3-NBN. The reproducibility of MAI was also analyzed relative to ESI and MALDI on three different mass spectrometers; ESI and MALDI equipped SYNAPT G2 mass spectrometers and an ESI equipped LTQ Velos mass spectrometer (Figure 3). The results obtained using MAI on each of these instruments were then compared to those obtained using ESI and MALDI. The RSD results obtained using ESI on the SYNAPT G2 and LTQ Velos were comparable and in the range of ca. 4-12% using 15 replicates. Similarly, the RSD values of MALDI measurements, obtained on the SYNAPT G2, ranged from 3.8-16% also using 15 replicates. The RSD values for MAI obtained using the same samples and number of replicates on the atmospheric pressure SYNAPT G2 were in the range of 2.4-9.9% and on the LTQ Velos 3.1-4.6%, while MAI measurements taken on the intermediate pressure source of the SYNAPT G2 ranged from 3.8-20% (Supporting Information Table S8). The wider range of RSD values for MAI obtained on the intermediate pressure source of the SYNAPT G2 may be due to sublimation beginning as soon as the plate is loaded, well before data acquisition can be started. Based on these results, the reproducibility of MAI using the atmospheric pressure inlet for the mixture of drug compounds was comparable to ESI on both mass spectrometers and an improvement over MALDI on the SYNAPT G2. MAI and MALDI using the intermediate pressure source of the SYNAPT G2 gave roughly equivalent results.



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We also compared the consumption of 1 µL of the same spiked urine sample among ionization methods (Supporting Information Figure S4). MAI readily detected all six drugs in good abundance using the matrix 3-NBN. Sprayable conditions were obtained by diluting 1 µL of the 2 ppm sample to 20 µL prior to analysis by ESI. All six drugs were detected in the summed mass spectrum, but in much lower abundance and with an increase in background noise. Using the previous MALDI conditions and matrix CHCA, the undiluted urine sample was analyzed by MALDI. Ablating the entire matrix spot containing 1 µL of analyte, excellent ion abundance was achieved for buspirone and clozapine, but morphine, codeine, oxymorphone and oxycodone are less abundant than the surrounding background noise. Even from undiluted urine samples, MAI ionizes drugs reproducibly and with good signal to noise.

Quantification Study of Illicit Drugs from Human Urine In order to quantitatively analyze an infant urine sample, a MAI-MS method was developed using the mixture of six drug standards and their respective internal standards spiked into a sample of blank human urine. The reproducibility of these drugs was determined from the spiked urine sample on both the SYNAPT G2 and LTQ Velos mass spectrometers. Fifteen averaged acquisitions were used to calculate the RSD values that ranged from 3.4-29% on the SYNAPT G2 and 5.3-16% on the LTQ Velos (Supporting Information Figure S5, Supporting Information Table S9). The RSD values from the urine samples are greater than those of the standard aqueous methanol solutions, as may be expected with significantly more complex samples, but still fall within an acceptable range.


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The analytical performance of MAI was further analyzed by the linearity of calibration curves and the calculated limits of detection (LOD) of drugs spiked in urine. The R2 values were determined to be ≥ 0.99 for all drugs on the SYNAPT G2 and the LTQ Velos (Supporting Information Figure S6 and S7). The ranges of LOD obtained for the drugs spiked in urine, were 0.28-8.2 ppb and 0.12-3.9 ppb on the SYNAPT G2 and the LTQ Velos, respectively (Supporting Information Table S10). Because of the complexity of the urine sample, contamination effects were analyzed using a 1 pmol µL-1 standard of ubiquitin dissolved in aqueous methanol with 1% acetic acid. The ion abundance of this standard, measured in triplicate, was observed before, throughout, and after 200 drug spiked urine sample acquisitions. For this study, source temperatures of 30 °C and 150 °C were used on the SYNAPT G2 and LTQ Velos, respectively. The resulting spectra showed a relatively small decrease in ion abundance over the course of 200 injections (Supporting Information Figure S8 and S9). The developed method was sufficient for quantification of structurally distinct illicit drugs (Scheme 1.I) having different molecular weights, including directly from a drug spiked urine sample, (Supporting Information Figure S10). However, small molecules with closely related molecular weights such as isobars, or isomers, are generally differentiated by their retention time and/or fragmentation patterns using LC-MS/MS.29-33 MS/MS of codeine and hydrocodone, structurally similar drugs (Scheme 1.I.C and E.), have essentially the same fragmentation patterns, in terms of mass-to-charge (m/z) ratios, of the fragment ions. However, the ion abundances of the fragment ions differ



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significantly for the two opioids, as seen for m/z 199.09 in the Supporting Information (Figure S11), which allows differentiation of these species. A sample of human infant urine (OP1) from a patient whose mother had taken opioids during pregnancy was tested using MAI-MS and MS/MS. The sample showed a prominent signal at m/z 300 (Figure 4.I.A), which was suspected to be either codeine or hydrocodone. The protonated molecular ion at m/z 300 was fragmented by CID and compared to a solution of OP1 spiked with hydrocodone (2.5 ppm) (Supporting Information Figure S12). No significant differences were observed in the fragmentation pattern of the authentic hydrocodone sample was observed (Figure 4.I.B).

Had

codeine been present, the prominent fragment ion at m/z 199.09 would be expected to be diminished, thus confirming the presence of hydrocodone alone. The concentration of hydrocodone in the urine sample OP1 was quantified without prior sample purification or dilution by using the standard addition method with hydrocodone-D6 (1 ppm) as an internal standard. A linear regression calibration curve (Figure 4.II) was created from the mean values of five measurements at five concentrations of hydrocodone spiked into aliquots of OP1, resulting in a calculated hydrocodone concentration in the pure sample of 4.3 ppm after dilution correction. The regression line showed high linearity (R2=0.9958) without observable interference from the complex sample environment. The RSD was found to be in the range of 2.2-5.4%, which is notably better than the typically required 20% for clinical applications.28 These results are in excellent agreement using traditional methods (LC-MS) performed in the Detroit Medical Center on the same sample. An advantage of MAI-MS is that it nearly instantaneously detects a wide array of drugs simultaneously compared


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to targeted clinical approaches. 34 The OP1 sample contained a relatively high concentration (4.32 ppm) of hydrocodone. Future studies will expand on the quantification of lower concentrations of opioids (ppb) often seen in the clinical field.35

Conclusion MAI is a simple, rapid, and robust method that can reproducibly ionize samples even from complex biological matrices with minimal sample preparation. The reproducibility is sufficient for quantification using internal standards. This was validated on two different instruments, on separate days, and using different matrices. In addition, the reproducibility was found to be comparable with ESI and an improvement over MALDI. Analytes of interest can be quantified in lower ppm levels using MAI-MS and MS/MS directly from urine without dilution or sample treatment. With further engineering and method development, it seems reasonable that increased reproducibility, sensitivity, and speed of analyses will be obtainable, making MAI increasingly viable22,27 for use in clinical laboratories.



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Acknowledgements The authors are grateful for support from NSF STTR-1417124 and CHE-1411376, DuPont Young Professor Award, Eli Lilly Young Investigator Award, Waters Center of Innovation Award, WSU Schaap Faculty Award to ST, and DAAD PROMOS Scholarship to KJ. Special thanks to Professor Charles N. McEwen (MSTM and University of the Sciences) for his critical comments during the preparation of this work.


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Legends Scheme 1: Structures and respective molecular weights (MW) of (I.) drugs: A. morphine, B. oxymorphone, C. codeine, D. oxycodone, E. hydrocodone, G. buspirone, (-)-∆9-tetrahydrocannabinol, I. (±)-11-nor-9-carboxy-∆9F. clozapine, H. tetrahydrocannabinol; and (II.) matrices: A. 3-NBN (R1=CN), B. 2-NBN (R2=CN), C. methyl 2-methyl-3-nitrobenzoate, D. methyl 5-nitro-2-furoate, E. CHCA. Figure 1: Relative standard deviation of MAI-MS analyses of the 2 ppm drug mixture over five different days using matrix 3-NBN with the source temperature set to (A) 30 °C on a Waters SYNAPT G2 and (B) 150 °C on a Thermo LTQ Velos. Figure 2: Relative standard deviation of MAI-MS analyses of the 2 ppm drug mixture using matrices 3-NBN, 2-NBN, methyl 2-methyl-3-nitrobenzoate and methyl 5-nitro-2furoate (Scheme 1.II.) at (A) 30 °C on a Waters SYNAPT G2 and (B) 150 °C on a Thermo LTQ Velos. Figure 3: Relative standard deviation of the 2 ppm drug mixture using different ionization methods and mass spectrometers. MAI was performed on the atmospheric pressure (AP) source of a Waters SYNAPT G2, the AP source of a Thermo LTQ Velos and the intermediate pressure source of a Waters SYNAPT G2 using matrix 3-NBN. ESI measurements were performed on the atmospheric pressure sources of a Waters SYNAPT G2 and a Thermo LTQ Velos. MALDI matrix CHCA was used for all MALDI measurements on the intermediate pressure source of a Waters SYNAPT G2. Figure 4: Quantification of an illicit drug using MAI of undiluted infant urine: (I.A) A nontargeted full range mass spectrum. (I.B) CID MS/MS of molecular ion m/z 300 and comparison of fragmentation pattern taking into account m/z and ion intensity values (Supplemental Figure S12) for the characterization of hydrocodone. The fragment ion with the most suggestive intensity ratio for hydrocodone is indicated. (II.) Linear regression calibration and after dilution correction to quantify hydrocodone as present at 4.32 ppm. All analyses were performed using a Waters SYNAPT G2 with the Z-Spray source block temperature set to 30 °C.



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Schemes Scheme 1. I. A.

B.

285.34

C.

301.34

E.

D.

299.37

F.

315.36 G.

299.37

385.50

H.

I.

315.47 II. A. B.

148.12

344.45

C.

D.

195.17

326.82 E.

171.11

189.17


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Figures

Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.


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