A Study of Opiate, Opiate Metabolites and Antihistamines in Urine after Consumption of Cold Syrups by LC-MS/MS

Studying the origin of opiate and/or opiate metabolites in individual urine specimens after consumption of cold syrups is vital for patients, doctors, and law enforcement. A rapid liquid chromatography–tandem mass spectrometry method using “dilute-and-shoot” analysis without the need for extraction, hydrolysis and/or derivatization has been developed and validated. The approach provides linear ranges of 2.5–1000 ng mL−1 for 6-acetylmorphine, codeine, chlorpheniramine, and carbinoxamine, 2.5–800 ng mL−1 for morphine and morphine-3-β-d-glucuronide, and 2.5–600 ng mL−1 for morphine-6-β-d-glucuronide and codeine-6-β-d-glucuronide, with excellent correlation coefficients (R2 > 0.995) and matrix effects (< 5%). Urine samples collected from the ten participants orally administered cold syrups were analyzed. The results concluded that participants consuming codeine-containing cold syrups did not routinely pass urine tests for opiates, and their morphine–codeine concentration ratios (M/C) were not always < 1. In addition, the distribution map of the clinical total concentration of the sum of morphine and codeine against the antihistamines (chlorpheniramine or carbinoxamine) were plotted for discrimination of people who used cold syrups. The 15 real cases have been studied by using M/C rule, cutoff value, and distribution map, further revealing a potential approach to determine opiate metabolite in urine originating from cold syrups.


Introduction
Opiate metabolites usually refer to the metabolites of morphine, codeine, and heroin. Morphine and codeine are natural substances present in poppies, whereas heroin is a semisynthetic product derived from morphine. Heroin, considered one of the most serious drugs abused worldwide, has led to numerous deaths-approximately 15,482 in 2017 [1]. In forensic science, several analytical methods, including gas chromatography-mass spectrometry (GC-MS) [2,3], GC-tandem MS (GC-MS/MS) [4,5], and liquid chromatography-mass spectrometry (LC-MS/MS) [6,7], have been adapted for the identification of heroin, other opiates, and their metabolites.

Method Development and Assay Validation
Table S1 summarized the transitions, fragments, and collision energies for M3G, morphine-d 6 , morphine, M6G, C6G, codeine-d 6 , codeine, 6-AM, chlorpheniramine-d 6 , chlorpheniramine, and carbinoxamine optimized with an Agilent MassHunter optimizer. Figure S1 showed an example of the successful separation of each analyte (1000 ng mL −1 ) under the optimized conditions using LC-MS/MS in extracted-ion mode, and the retention times of the analytes were in the range of 2.8-10 min.
The correlation coefficients (R 2 ), calibrated linear ranges, limits of detections (LODs), carryovers, accuracies, and coefficients of variation (CV% values) for morphine, 6-AM, M3G, M6G, codeine, C6G, chlorpheniramine, and carbinoxamine were summarized in Table 1. All the calibration curves of the analytes were linear (R 2 > 0.995) for quantitation in different linear ranges. The calibrated linear ranges were 2.5-800 ng mL −1 for morphine and M3G, 2.5-600 ng mL −1 for M6G and C6G, and 2.5-1000 ng mL −1 for 6-AM, codeine, chlorpheniramine, and carbinoxamine. The LODs of the analytes were 0.2-1.3 ng mL −1 , confirming that the LC-MS/MS approach was sensitive for the quantitation of these analytes in urine samples. The linear ranges of morphine, M3G, M6G, and C6G, which contain phenol or glucuronide groups, resulting in pH-dependent ionization efficiency, were narrower than those of 6-AM, codeine, chlorpheniramine, and carbinoxamine. The LOD of morphine was higher than those of the other analytes, mainly because electrospray formation was hampered attributed to a lower content of organic modifier (water/acetonitrile = 83/17, v/v) at the retention time (3.7 min). The selectivity of this method and the influence of endogenous compounds were assessed using blank urine samples. As shown in Figure S2, unexpected interferences were not observed in the chromatograms, but obvious signals of the internal standards (ISs) and the analytes at their LODs were visible, confirming the excellent selectivity and sensitivity of the method [27][28][29][30]. Carryover was tested with the use of equation (1). The carryover indices of all analytes were within 1%, indicating the absence of significant carryover between the analyses of the working solutions at the highest and lowest tested concentrations [31]. The intraday assay precision values at low, moderate, and high concentrations of the testing analytes were 0.2%-3.6%, 0.2%-3.3%, and 0.7%-3.1%, respectively. The interday assay precision values at low, moderate, and high concentrations were 1.8%-4.2%, 1.4%-8.3%, and 0.9%-6.2% at 0, 7, and 14 days, respectively. The intraday and interday assay accuracies were in the range of 83.1% to 112.2% and 84.1% to 104.6% (i.e., within the ±20% maximum acceptance criteria) [32]. Table S2 listed the slopes and matrix effects (MEs) of the urine matrix calibration curves obtained using "dilute-and-shoot" analysis without extraction, hydrolysis and/or derivatization. The MEs were assessed by analyzing the analytes spiked into five individual blank urine samples and pure solvent according to a previously reported procedure [33,34]. Morphine, 6-AM, M6G, codeine, C6G, and carbinoxamine demonstrated MEs of 0% to 8.7%, implying that the urine matrix did not significantly affect the ionization of these analytes (within −15% to 15%) [33,35]. However, the urine matrix significantly enhanced the signals of M3G and chlorpheniramine (MEs > 15%). The highest matrix effect (approximately 32%) for M3G was observed, mainly because that higher amounts of polar constituents from urine matrix were co-eluted with M3G for ion enhancement at a short retention time (2.8 min) in a higher content of aqueous modifier (water/acetonitrile = 91/9, v/v) [36][37][38]. Thus, to reduce this effect, a matrix-match method was used for quantitation. As expected, a high precision (<5%) of MEs was achieved in the five individual blank urine samples. The interference effects were assessed by analyzing our targets when mixed with high concentrations of the interferents. The results revealed biases in the range of −4% to 9% within ±20% of the target concentration [39]. The dilution integrity was assessed for 2-, 10-and 20-fold dilutions as shown in Table S3. The results revealed that the accuracies were in the range of 94.0% to 106.9%, which are within the acceptable variation of ±15% of the nominal concentration, and precisions were in the range of 0.2% to 5.2% in the three replicates. Table S4 showed the excellent stabilities of the analytes during storage, the concentrations of all the analytes were stable within a variation of ±10% over a period of 28 days, indicating that urine samples stored at 4 • C prior to analysis were suitable for these assays.

Clinical Trials Study
Cold syrups were orally administered to ten participants following the suggested use on packaging (4.8 or 5 mg of codeine phosphate in each dose, three times a day for three days). Three different cold syrups were tested, and the average analyte concentrations and the calculated total concentration of morphine or codeine in the urine samples from the ten participants were displayed in Figure 1. For the samples (Consrine, ssBuron, and Pabron), the average concentrations of C6G (419-1190, 624-1476, and 358-1406 ng mL −1 ) and M3G (177-353, 326-508, and 114-263 ng mL −1 ) were obviously higher than those of codeine (249-940, 307-944, and 226-728 ng mL −1 ) and morphine (7.6-130, 8.7-17, and 8.2-13 ng mL −1 ), respectively, during the trial period of 0-66 h, indicating that opiate-glucuronides were the major metabolites of the opiate. When syrup consumption was terminated, the concentrations of the opiate and the opiate metabolites decreased quickly. On the other hand, chlorpheniramine or carbinoxamine was still detectable after 84-132 h of administration, and thus these compounds can be used as markers to determine the use of cold syrups. The changes in opiate concentrations as a function of time were slightly different for the three formulations due to differential lifestyles and metabolic rates of the participants, the times at which the urine samples were collected, and the small number of samples. A time interval of cold syrups taken by participants for their last dose that day until the next day's use was approximately 12 h, which caused decreasing concentrations of each metabolite in the urine samples under 30-48 h of administration.
The details of the total concentrations of codeine or morphine in the urine samples of all participants were displayed in Figure Figure 3. The results showed that the M/C values were under 1 during the trial period of 0-66 h, which was consistent with the rule of M/C < 1 [21]. Unfortunately, after 72 h of administration, the M/C values increased gradually, eventually violating the rule of M/C < 1, attributed to that the metabolite rate of morphine was slower than that of codeine [40].    Table 2 listed the concentrations of the 6-AM, opiate, opiate metabolites, and M/C values in samples from 15 real cases. A worldwide-accepted biomarker, 6-AM, is used to determine suspects for their heroin consumption. The case 1, 6, 13, and 15 found 6-AM in their urine samples, confirming those suspects for heroin use. However, 6-AM was not detected in other cases, mainly because those suspects probably did not consume heroin, or the short life of 6-AM was metabolized to morphine in bodies after heroin consumption. In addition, the M/C values and the concentrations of morphine or codeine from samples 1-8 were over 5 and over the cutoff value of 300 ng mL −1 (for Taiwan), respectively. Thus, we can deduce that these people did abuse heroin [20]. Interestingly, because chlorpheniramine was detected in cases 9-15, we can infer that these suspects consumed cold syrups or other chlorpheniramine-containing medicines. The M/C values from samples 9 and 10 were both within 1. As for sample 11, codeine or its metabolites were not detected, thus its M/C value referred to as infinity was close to those of clinical trial results during the trial period of 108-132 h, due to morphine being eliminated more slowly than codeine in the body [40]. The three cases (case 9-11) passing opiate urine test were not prosecuted because of their concentrations of opiate metabolites under the cutoff value (300 ng mL −1 ). On the other hand, the cases 12-15 with their M/C values over 3 as well as their concentrations of opiate metabolites over the cutoff value were all prosecuted. Based on our practical experience, cold syrups or other morphine/codeine-containing medicines were likely consumed to mask the illegal abuse of heroin, allowing the "poppy seed defense" for suspects in cases 12-15. Furthermore, the distribution of total concentration of the sum of morphine and codeine against chlorpheniramine or carbinoxamine obtained from clinical trials, as well as those from samples 9-15 were mapped in Figure 4A,B. The results showed that distributions of samples 9-11 were overlapped with those determined in the clinical trials, confirming the suspects in the three cases consumed cold syrups containing codeine and chlorpheniramine. Conversely, samples 12-15 have no overlapping with those acquired from clinical trials, further revealing the four suspects consumed heroin and cold syrups. Finally, the suspects in cases 1-8 and 12-15 were prosecuted and thus judged in a court in Taiwan to have consumed heroin. Therefore, this distribution map showed the potential for determining opiate and opiate metabolites derived from cold syrups in urine samples. However, it was noted that for cancer patients, morphine was used for the treatment of cancer pain [41,42]. It was still difficult to discriminate against the opiate metabolites in urine from heroin or morphine without detection of 6-AM, but patients can provide certificates of diagnosis and prescriptions from doctors to prove their legal use.

Clinical Trial Urine Samples
Ten participants (5 male and 5 female) aged 22-56 years with a mean weight of 67 kg were administered 10 mL of cold syrup after each meal three times a day for three days (72 h) according to suggested use described by the manufacturer. Urine samples were collected before and after oral administration of the cold syrup (i.e., before administration and 6, 18, 30, 42, 54, 66, 84, 108, and 132 h after administration), and the samples were stored at 4 • C until analysis. All participants agreed this study and gave their informed consents before experiments. All experiments were performed in compliance with the ICH E6 Guidance for Industry (E6 Good Clinical Practice: Consolidated Guidance) and approved by the Joint Institutional Review Board of Taiwan (Certificate of Approval JIRB No: 18-S-004-1).

Urine Samples from Real Cases
Urine samples from 15 suspects in real cases involving opiate abuse were acquired from the Taipei District Prosecutors Office in Taiwan and were used for analysis. All urine samples were stored at 4 • C until analysis. According to the Court's Judgments, 12 of the suspects were convicted of heroin consumption, and the others were found innocent.

Apparatus
The LC-MS/MS system consisted of an autosampler (Agilent G7167A, Santa Clara, USA), a binary pump (Agilent G7112B, Santa Clara, USA), a separation column (3.0 × 100 mm, 2.7 µm, Poroshell 120SB-AQ Agilent, Santa Clara, USA), and a mass spectrometer (Agilent 6470 QqQ-MS, Santa Clara, USA), and the urine samples were assessed in electrospray ionization mode. Mass spectrometry was performed using multiple reaction monitoring (MRM) mode to analyze 8 species, including morphine, 6-AM, M3G, M6G, codeine, C6G, chlorpheniramine, and carbinoxamine. Morphine-d 6 , codeine-d 6 , and chlorpheniramine-d 6 were used as internal standards. The LC-MS/MS system was operated under the following conditions: drying gas temperature of 280 • C, flow rate of 8 L min −1 , and nebulizer pressure of 45 psi. Two product ions were obtained for each analyte, and their MRM ion ratios calculated from the peak areas of the two product ions were used for qualitative evaluation, the fragment with the greater area was used for quantitation. The mobile phase was a mixture of solvent A (0.1% formic acid aqueous solution) and solvent B (100% acetonitrile) at a flow rate of 0.3 mL min −1 . The gradient program was set as follows: 0-0.5 min (a volume ratio of solvent A to B of 97:3), 0.5-3 min (decreasing to 90:10), 3-5 min (decreasing to 70:30), 5-10 min (decreasing to 50:50), 10-15 min (decreasing to 20:80), 15-17 min (decreasing to 0:100), 17-18 min (0:100), and 18-28 min (97:3). The sample was injected using an autosampler, and the injection volume was 5 µL. The Software (Version B.08.00, Agilent Technologies Inc., 2016) was used for quantitative and qualitative analyses.

Analytical Strategy
According to the guideline of SWGTOX Standard Practices for Method Validation, the US Food and Drug Administration (FDA), and the European Medicines Agency (EMA) Guidelines on Bioanalytical Method [32,43,44], the parameters that must be validated include the dynamic range, LOQ, selectivity, precision, accuracy, carryover effect, matrix effect, interferents, dilution integrity (dilution factor: 2, 10, and 20), and stability. A rapid "dilute-and-shoot" analytical method was validated and used to determine the concentrations of opiate, opiate metabolites, chlorpheniramine, and carbinoxamine in urine samples from participants that had been orally administered cold syrup and from 15 people suspected of abusing opiates.

Dynamic Ranges, LOQs, and LODs
Aliquots (20 µL) of the working solution, blank urine (20 µL), and mixed IS solution (20 µL) were mixed with 940 µL of the eluent (A: B = 97: 3) in 2-mL vials to prepare the samples for calibration (n = 5). A weighted (1/x) regression model was used to prepare calibration curves based on the peak area ratios of the analytes relative to the ISs. The back-calculated concentrations of the calibrators are within ±15% of the target values at all points except the LOQ. The lowest concentration of each analyte in the linear range was defined as the LOQ, and at this concentration, the calculated value was within ±20%. In addition, to determine the LOD, the most dilute working solution was diluted 1 to 10 times with a 1:1 (v/v) mixture of methanol and water. Aliquots (20 µL) of the aforementioned diluted working solutions, 20 µL of blank urine, and 20 µL of the mixed IS solution were mixed with 940 µL of the eluent (A: B = 97:3) in 2-mL vials to prepare the samples for qualitative analysis. The LOD of each analyte was set as the concentration producing a signal with an intensity 3 times that of the noise (S/N = 3, n = 7).

Selectivity
The selectivity of the analytic method was assessed by comparing the chromatograms of ten lots of blank urine, blank urine spiked with the analytes at their LOQs, and IS-spiked blank urine. Each

Accuracy and Precision
The recovery percentage from the nominal concentration and CV % from interday and intraday assays based on analysis of QC samples (n = 6) were used to assess the accuracy and precision of this method. QC samples were prepared by mixing 20 µL of QC solutions, 20 µL of blank urine, and 20 µL of IS mixture solution with 940 µL of the eluent (A: B = 97:3) in 2-mL vials.

Carryover
The carryover was assessed by analyzing the most dilute working solution four times immediately after analyzing the most concentrated working solution three times. The carryover index was calculated as follows [31]: where L 1 , L 3 , and L 4 are the peak areas of the most dilute working solutions from runs 1, 3, and 4, respectively, and H 2 and H 3 are the peak areas of the most concentrated working solutions from runs 2 and 3, respectively.

Matrix Effect
MEs were investigated according to the literature [30,31]. Interference samples were prepared by mixing our target analytes with high concentrations (1000 ng mL −1 ) of likely interferents. These samples were analyzed in three replicates and then quantitatively evaluated for interference effects.

Conclusions
A rapid analytic method for the quantitation of opiate, opiate metabolites, and antihistamines (chlorpheniramine and carbinoxamine) in urine through LC-MS/MS was developed and validated. The results of a clinical trial revealed that people consuming cold syrups do not consistently pass opiate urine tests and the samples do not have morphine-codeine concentration ratios < 1 routinely. The distribution map of the total concentration of the sum of morphine and codeine against chlorpheniramine or carbinoxamine in the urine samples from participants were plotted, respectively, which were used to determine opiate metabolites in urine originated from cold syrups. Samples from fifteen people suspected of heroin abuse were analyzed and validated using M/C rule, cutoff value, and distribution map. To the best of our knowledge, this is the first report to discuss the concentrations of antihistamines and to study the distribution of total concentration of the sum of morphine and codeine against an antihistamine derived from cold syrup use.
Supplementary Materials: The following are available online, Table S1: MRM transitions, retention times, and tuning parameters of the analytes, Table S2: Slopes and MEs of urine matrix calibration curves using "dilute-and-shoot" analyses, Table S3: Accuracy and precision for QC in dilution integrity test, Table S4: Stability assessment, Figure S1: Chromatograms of analytes obtained using LC-MS/MS in extracted-ion mode, Figure S2: Chromatograms of ten lots of blank urine and samples spiked with IS or LOQ-analytes in total-ion mode.