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Paper Spray Ionization Coupled to High Resolution Tandem Mass Spectrometry for Comprehensive Urine Drug Testing in Comparison to Liquid Chromatography-coupled Techniques after Urine Precipitation or Dried Urine Spot Workup Julian A. Michely, Markus R. Meyer, and Hans H. Maurer Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03398 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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Analytical Chemistry

Paper Spray Ionization Coupled to High Resolution Tandem Mass Spectrometry

for

Comprehensive

Urine

Drug

Testing

in

Comparison to Liquid Chromatography-coupled Techniques after Urine Precipitation or Dried Urine Spot Workup

Julian A. Michely, Markus R. Meyer, and Hans H. Maurer*

Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, D-66421 Homburg (Saar), Germany

ACS Paragon Plus Environment

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ABSTRACT: Screening procedures using high resolution (HR)-mass spectrometry (MS) are getting more and more important e.g. for drug testing or adherence monitoring. Approaches usually include time-consuming sample preparation and compound separation by liquid chromatography (LC). The paper spray ionization (PSI) technique coupled to MS might overcome these steps by direct analysis of complex mixtures without extraction and separation. In recent years, this technology proved its potential for quantification and/or qualitative screening in biofluids. However, so far PSI-MS was only applied to procedures covering a limited number of targets. Therefore, a PSI-HR-MS/MS approach was developed and successfully validated for comprehensive urine screening. The procedure showed high matrix effects for most drugs but still acceptable limits of identification. Applicability was tested by analyses of three proficiency tests for systematic toxicological analysis and of 103 authentic human urine samples. Its screening power was compared to that of published LC-HR-MS/MS procedures after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup by conjugate cleavage and liquid extraction (DUSglucE). In the authentic samples, 73% of all 777 drug intakes were detectable with the new approach. The LC-HR-MS/MS screening approaches detected more drugs, particularly in samples with low analyte concentrations, with values of 88% after UglucP or 76% after DUSglucE. In conclusion, the new PSI-HR-MS/MS screening approach was suitable for comprehensive urine screening of different drug classes and might be a promising alternative to conventional procedures. However, limitations should be considered such as detection of drugs in low concentrations and risk of false positive or negative results caused by mixed spectra.


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Mass spectrometry (MS)-based screening procedures deliver reliable data for drug testing, adherence monitoring, or in poisoning cases e.g. in clinical and forensic toxicology.1 In recent years, more and more screening approaches used high resolution (HR) techniques and took advantage of their high selectivity and sensitivity.2-8 Most of these approaches included an appropriate sample preparation and compound separation by liquid chromatography (LC) resulting in high sensitivity and robustness, but with prolonged turnaround time. Paper spray ionization (PSI) as an ambient ionization technique should allow reducing workload and analysis time in (urine) drug testing. It should also be suitable as a sampling strategy by reducing sample transport and storage costs and extent analyte stability due to its dried form. In 2010 Wang et al.9 showed the potential of PSI-MS as alternative for direct analysis of complex mixtures. They used a triangular piece of paper, which was spiked with the sample material and positioned in front of the MS source. Spray generation as well as analyte transport and ionization was achieved by wetting the paper with a solvent and subsequently applying voltage.9 In the following years, the advantages of PSI coupled to (HR-)MS were addressed in many publications for quantification and/or qualitative screening of e.g. therapeutic or illicit drugs in matrices such as whole blood or urine as well as e.g. (toxic) contaminants in food.10-26 A method overview by Klampfl and Himmelsbach10 in 2015 described PSI-MS analysis as the most popular direct ionization technique meanwhile. However, so far PSI-MS was only applied to screening and/or quantification procedures, which covered limited numbers of targets in blood such as the drugs of abuse cocaine, heroin, methamphetamine, oxycodone, and buprenorphine17 or amphetamine, methamphetamine,

3,4-methylenedioxyamphetamine

(MDA),

3,4-methylenedioxy-N-

methylamphetamine (MDMA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), morphine, cocaine, and ∆9-tetrahydrocannabinol (THC)22, respectively. A comprehensive urine screening


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covering a broad range of targets is still missing. It should also include the drug metabolites as they are often the major or even unique target in urine.8,27 Therefore, a common PSI setup coupled to HR-MS/MS was modified for a metabolite-based comprehensive urine screening approach and then validated including matrix effects, ionization effects of co-eluting analytes, and cartridge reanalyzability. Furthermore, its screening power was tested in comparison to published LC-HR-MS/MS procedures8 that already showed their high sensitivity for broad screening in previous studies.28,29 For all approaches, the identical reference library was used for compound identification covering more than 5,000 spectra of parent compounds as well as their phase I and II metabolites.30

EXPERIMENTAL SECTION Chemicals and Materials. Velox Sample Cartridges were provided by Prosolia (Indianapolis, USA). BG100 ß-glucuronidase solution (100,000 units/mL) from genetically selected Haliotis rufescens was purchased from KURA Biotec (Puerto Varas, Chile), acetic acid, amiodarone, ammonium acetate, ammonium carbonate, ammonium formate, bisoprolol, buprenorphine-3-glucuronide, clonidine, codeine-6-glucuronide, diclofenac, diphenhydramine, flufenamic acid, formic acid, levetiracetam, lorazepam glucuronide, losartan, morphine-3glucuronide,

oxazepam

glucuronide,

pantoprazole,

paracetamol

sulfate,

pentobarbital,

promethazine, quetiapine, raloxifene-6-glucuronide, risperidone, sertraline, and thiopental from Sigma-Aldrich (Taufkirchen, Germany), amitriptyline, amphetamine, codeine, diazepam, and morphine from LGC Standards (Wesel, Germany), acetonitrile, methanol, water, as well as all other chemicals from VWR (Darmstadt, Germany). All chemicals were of analytical grade or better.


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Urine Samples. Certified proficiency test urine samples for qualitative systematic toxicological analysis were purchased from RfB (Referenzinstitut fuer Bioanalytik, Bonn, Germany). Blank urine samples were collected from healthy volunteers and authentic urine samples submitted to the authors’ laboratory for toxicological diagnostics. The authentic human urine samples were identical to those used for the previous LC-HR-MS/MS study and were submitted in context of drug testing or adherence monitoring.29 Sample Preparation. A volume of 90 µL urine was mixed with 10 µL of a ß-glucuronidase solution in 100 mM ammonium acetate (1:1), resulting in 5,000 units/mL at pH 5.2 in analogy to a published procedure.31 Conjugate cleavage was performed at 55°C for 30 min followed by cooling at 4°C for 15 min and centrifugation (10,000 g, 2 min). A 20-µL aliquot was then spotted onto Velox Sample Cartridges and dried at room temperature for about 15 min. PSI-HR-MS/MS Apparatus and Conditions. The prepared cartridges were directly analyzed using an automated Velox 360 paper spray autosampler (Prosolia) coupled to a QExactive Focus HR-MS/MS system (Thermo Fisher Scientific, San Jose, USA). Mass calibration was done prior to analysis according to the manufacturer’s recommendations every 72 hours using external mass calibration. For elution and transfer of the compounds into the apparatus, a solvent mixture of acetonitrile:water:formic acid (90:10:0.1, v:v:v) was used. It was applied to the cartridge with following settings: sample dispense pump (on top of the sample spot), A; number of injections, 5 times with 3 µL; pump A sample dispense delay (between injections), 2 s; cartridge dispense pump (behind the sample spot), B; number of injections, 12 times with 10 µL; pump B cartridge dispense delay, 5 s; and MS acquisition time, 4 min. The sample and cartridge dispenses of the samples were performed during the MS acquisition of the previous samples, leading to a total run time of approximately 4 min per sample except for the first one.


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The PSI source conditions were as follows: distance between paper tip and MS, 5 mm; spray voltage, 0 kV from 0.0-0.1 min and 3.9-4.0 min, +5.00 kV (positive) or -5.00 kV (negative) from 0.1-3.9 min; capillary temperature, 300°C; and S-lens RF level, 60.0. MS conditions were those described in previous studies8,29 performing in full scan mode and subsequent data dependent acquisition (DDA) with minor modifications for PSI analysis. The settings for the full scan mode were as follows: polarity, switching; resolution, 35,000; scan range, m/z 130-1000; automatic gain control (AGC) target, 1e6; maximum injection time, 120 ms; microscans, 1; and spectrum data type, profile. The settings for the DDA mode were as follows: dd-MS2, discovery; resolution, 17,500; isolation window, m/z 1.0; high collision dissociation (HCD) cell stepped normalized collision energy (NCE), 17.5, 35.0, and 52.5%; AGC target, 2e5; maximum injection time, 250 ms; loop count, 3; minimum AGC target, 2.5e3 (corresponds to a signal intensity threshold of 1e4); dynamic exclusion, 120.0 s with ±400 milli mass units (mmu) mass tolerance; exclude isotopes, on; and spectrum data type, profile. For focused screening, the addition of a parent reaction monitoring (PRM) mode was performed from 2.0-3.0 min on the targets of interest defined by an inclusion list. Xcalibur 4.0 software (Thermo Fisher Scientific) was used for data handling. For data evaluation of the obtained chronograms (corresponding to chromatograms after chromatographic separation), TraceFinder Clinical Research 4.1 software (Thermo Fisher Scientific) was used in analogy to previous studies.8,29 The settings for processing were as follows: retention time limits, 0.1-3.9 min; precursor peak detection in full scan, enabled; threshold override, 5,000; signal to noise (S/N) ratio threshold, 1; mass tolerance, 500 parts per million (ppm; finally monitored mass tolerance of 10 ppm in the result report); fragment ions, identify; ignore if not defined, enabled; minimum number of fragments, 2; intensity threshold,


6

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100; mass tolerance, 10 ppm; isotopic pattern, disabled; library search, identify; search type, HighChem; score threshold, 30%, ignore precursor, disabled; and reverse search, disabled. The reference library was the metabolite-based Maurer/Meyer/Helfer/Weber (MMHW) library.30 Method Validation. According to international guidelines,32,33 the approach was validated with parameters and criteria for qualitative approaches. In analogy to a study described elsewhere,31 two methanolic stock solutions were prepared, containing a mixture of 20 drugs (mixture A) or 7 conjugates (mixture B) at concentrations of 50,000 ng/mL, respectively. These drugs covered a broad range of physicochemical properties. Mixture A: amiodarone, amitriptyline,

amphetamine,

diphenhydramine,

flufenamic

bisoprolol, acid,

clonidine,

levetiracetam,

codeine, losartan,

diazepam, morphine,

diclofenac, pantoprazole,

pentobarbital, promethazine, quetiapine, risperidone, sertraline, and thiopental. Mixture B: buprenorphine-3-glucuronide, codeine-6-glucuronide, lorazepam glucuronide, morphine-3glucuronide, oxazepam glucuronide, paracetamol sulfate, and raloxifene-6-glucuronide. Mixture A was used for determination of analyte carry-over, ionization effects of co-eluting analytes, matrix effect, and limit of identification (LOI) in analogy to published procedures8,31 and mixture B only for the determination of matrix effect. All drugs were monitored in positive mode, except for diclofenac, flufenamic acid, pentobarbital, and thiopental, which were monitored in negative mode. Selectivity. Interfering endogenous compounds were checked by ten individual blank human urine samples from different sources, which were prepared, analyzed, and evaluated as described above. Analyte Carry-over. Blank urine samples and urines spiked with mixture A to final drug concentrations of 50,000 ng/mL were prepared as described above in three replicates,


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respectively.31 For the latter, mixture A stock solution was evaporated at room temperature under nitrogen stream and reconstituted in one-tenth volume methanol before spiking. The analyses of the three spiked samples were followed by those of the three blank samples and the identified drugs monitored in the blank urine samples. Ionization Effects of Co-eluting Analytes. One sample set of blank urine spiked with mixture A to final drug concentrations of 1,000 ng/mL was prepared and analyzed in comparison to sample sets of blank urine spiked with the single drugs at identical concentrations, each in three replicates. The absolute areas of the exact protonated or deprotonated molecule masses (±5 ppm) were monitored in the corresponding reconstructed HR-MS ion chronograms and the ionization effects of co-eluting analytes calculated by comparing the sample set spiked with the mixture to those spiked with the single drugs. The criteria for ion suppression or ion enhancement were set to ±25% according to a published procedure.34 Matrix Effect. According to Matuszewski et al.,35 two sample sets were prepared. Sample set 1 consisted of neat methanolic solution and sample set 2 of urine from six different individuals, all containing mixture A or B in final drug concentrations of 1,000 ng/mL. The samples were prepared as described above, but without conjugate cleavage for those samples containing mixture B. They were analyzed in six replicates for sample set 1 and once per individual urine sample of sample set 2. Then, the absolute areas of the exact protonated or deprotonated molecule masses (±5 ppm) in the corresponding reconstructed HR-MS ion chronograms were monitored. For calculation of the matrix effect, areas from sample set 2 were compared to those of sample set 1. LOI Determination. Blank urine samples were spiked with mixture A to final drug concentrations of 10,000, 8,000, 6,000, 4,000, 2,000, 1,000, 800, 600, 400, 300, 200, 100, 80, 60,


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40, 20, 10, 5, or 1 ng/mL, each followed by preparation, analysis, and evaluation by the software. In addition to this general screening, a focused screening was performed with an inclusion list containing the target masses of mixture A. The LOI was defined as the concentration where a particular drug was still identified by the software with given criteria. Cartridge Reanalyzability. Mixture A was spiked in blank urine samples to final drug concentrations of 1,000 ng/mL and prepared in three replicates. Then, each cartridge was analyzed eight times with drying steps at room temperature in between. The absolute areas of the exact protonated or deprotonated molecule masses (±5 ppm) were monitored in the corresponding reconstructed HR-MS ion chronograms and calculated in relation to those of the first run, which was set to 100%. All runs were also reevaluated by the TraceFinder software for identification of eluted drugs. Applicability. Three proficiency tests for systematic toxicological analysis were prepared and analyzed in the PSI-HR-MS/MS screening approach as described above. In addition, 103 authentic human urine samples were prepared and analyzed by the PSI-HR-MS/MS screening approach as described above. The obtained results were compared to corresponding published screening data of LC-HR-MS/MS approaches using the identical mass spectrometer and data evaluation after urine precipitation with conjugate cleavage (UglucP) or as dried urine spot after on-spot cleavage followed by liquid extraction (DUSglucE).29 A urine sample was defined positive for a particular drug when at least the parent compound or one metabolite was detectable and a drug intake was defined as a positive urine sample for a particular drug in at least one screening approach.

RESULTS AND DISCUSSION


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Method Development and Optimization for PSI-HR-MS/MS. First, among the tested solvent compositions, acetonitrile:water (90:10, v:v) mixture was best for analyte elution and for producing a stable spray with long duration, continuous elution, and transfer of the compounds to the MS system. These findings were in line with Vega et al.36 who concluded high analyte signals and low ion suppressions using acetonitrile-based solvents for urine samples. Second, among the tested ionization modifier, addition of 0.1% formic acid was the best compromise for most of the tested drugs, in positive as well as in negative ionization mode. Furthermore, the time-limiting factor for signal duration seemed to be the solvent volume and not the amount of analyte that could be eluted from the paper. Therefore, a relatively high total solvent volume of 135 µL was used that could still be handled by the cartridge without dripping down. For continuous elution, it was beneficial to first dispense solvent on top of the sample spot (sample dispense) for rewetting the matrix and for avoiding an analyte focusing at the tip of the paper before dispensing behind the sample spot (cartridge dispense). With optimized settings, a stable spray could be formed for up to 4 min. However, a stronger retention on the paper was observed for compounds with hydrophilic properties resulting in a slight separation. This observation was in line with results described by Vega et al.,36 who revealed different elution properties for analytes or matrix components. The separation effect is exemplified in Figure 1 with reconstructed HR-MS ion chronograms of the exact protonated molecule masses (±5 ppm) of amiodarone, bisoprolol, morphine, and codeine-6-glucuronide. In addition, the logP values of the drugs are given.31 For the MS and data evaluation settings, few adoptions were done compared to the given reference.29 First, the dynamic exclusion time for the DDA-based trigger of the MS spectra was set to the middle of the chronogram (2 min). This setting allowed a reproducible and reliable


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detection of particularly hydrophilic drugs. Second, the mass tolerance for the dynamic exclusion was set to ±400 mmu due to the isolation window of m/z 1.0 of the quadrupole. This resulted in mixed spectra of simultaneously eluting compounds with identical nominal precursor masses. An example is depicted in Figure 2 showing the HR-MS/MS reference spectra30 of morphine (A) and of an endogenous biomolecule (B) as well as the measured mixed spectrum at a morphine concentration of 1,000 ng/mL in urine (C). In the mixed spectrum, morphine could be identified by the software via the low abundant, but specific fragment ions of m/z 125.0235 and 229.0857 and the precursor ion of m/z 286.1436. Third, the high mass tolerance in MS settings had to be considered in data evaluation settings for precursor detection in the used version of the TraceFinder software. This was necessary for reliable display of compounds even with accurate measured precursor masses of ±10 ppm deviation, which were monitored in the result report. Finally, the match threshold for the library score was lowered from 60 to 30% because of the above-mentioned mixed spectra. However, the risk of false positives must be considered. Method Validation. The tested mixtures covered several drug classes with a broad range of physicochemical properties. This strategy already showed its suitability for method validation of comprehensive screening procedures in previous studies.8,27,31 In selectivity testing, false positive amphetamine results were observed in three of ten tested blank human urine samples. They were probably caused by one or more co-eluting endogenous biomolecules, forming a spectrum with two fragment ions, which were of identical accurate masses compared to the main fragment ions of the amphetamine reference spectrum30 with its generally poor

fragmentation

pattern

after

electrospray ionization.4,30,37,38

Therefore,

amphetamine could only be considered as positive with at least three matched fragment ions and need to be confirmed in case of doubt. Thus, the number of false positives could be eliminated.


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However, compared to approaches including chromatographic separation, the risk of false positive and negative results is higher in PSI because of mixed spectra and lack of retention times as a further identification criterion. Therefore, critical manual inspection of the automatically obtained screening results was essential. The risk of analyte carry-over seemed to be rather low due to individual cartridges and no direct contact of any device parts to the sample.16,24 However, contaminations of the MS source could be a problem in such direct analysis procedures of raw samples.24 Therefore, the spiked urine samples were analyzed consecutively forcing analyte accumulation inside the MS source. In all three following blank urine samples, amphetamine, flufenamic acid, and levetiracetam were observed, pentobarbital in two of them, and risperidone in one of them. Thus, the tendency for carry-over seemed to be increased for hydrophilic compounds as most detected drugs showed logP values between -0.6 and 2.6.31 Only that of flufenamic acid was much higher with a logP of 5.3 (https://www.drugbank.ca). Therefore, high lipophilicity might also lead to compound carryover at least in combination with e.g. high ionization response or low ionization effects due to negative mode.8 For avoiding carry-over, at least three blank cartridges must be analyzed between two samples. However, this seemed not to be practical and represented a key issue of this approach. This problem should be solved before PSI implementation in routine analysis. When testing ionization effects of co-eluting analytes, all monitored analytes showed coelution because of almost no chromatographic separation. For assessing their effect on ion suppression or enhancement, urine samples containing mixture A were analyzed in comparison to spiked samples of single drugs in identical concentrations. A summary of the ionization effects of co-eluting analytes with their coefficient of variation (CV) is shown in Table 1. They were between -31 and +23% (±6-44%) for all drugs, whereby amphetamine, promethazine, and


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risperidone showed ion suppression considering the requested criteria. In conclusion, the ionization effects caused by co-eluting analytes seemed to be rather low considering the fact that almost every analyte co-eluted. The matrix effect with the coefficient of variation (CV) of urine samples containing mixture A are summarized in Table 1. For all these drugs, relatively high matrix effects with ion suppression were observed. They ranged between -70 to -98% (±2-24%) for all drugs monitored in positive mode, but only between -18 to -66% (±20-30%) for those monitored in negative mode. Such findings were also described by Vega et al.,36 who observed high matrix effects up to -90% in urine. For improving these suppressive effects, one possibility might be the modification of the cartridge paper surface to retain more matrix components. However, it should be mentioned that rather high matrix effects could be acceptable if the requested LOIs are achieved. For urine samples containing mixture B, all conjugates showed matrix effects of 100%. Despite the fact that these highly hydrophilic glucuronides or sulfates seemed to show severe adhesion to the paper with late elution in the matrix-free samples, spiked urine samples showed no signals at all. This could either be caused by high ion suppression or by adhesion enhancement due to the urine matrix. Conjugate cleavage, at least of the glucuronides, is therefore mandatory for reliable detection of drugs with high urinary conjugate excretion such as oxazepam or lorazepam.39 In Table 2, the determined LOIs of the drugs included in mixture A are summarized for general and focused screening. In addition, published LOIs by Helfer et al.8 using a LC-HRMS/MS approach after simple urine precipitation were included for comparison. Concerning the general screening, most drugs showed acceptable LOIs between 10-600 ng/mL, but also high LOIs of 10,000 ng/mL for levetiracetam, pentobarbital, and thiopental. Concerning drugs of


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abuse, the cut-off concentrations recommended by the European Laboratory Guidelines for Defensible Workplace Drug Testing40 were fulfilled for the tested drugs amphetamine, codeine, diazepam, and morphine. In comparison to the LC-HR-MS/MS screening approach after simple urine precipitation, four drugs showed identical, two lower, and eight higher LOIs. Six of the studied drugs were not included in the mentioned reference.8 In cases of drugs with higher LOIs, only bisoprolol showed a LOI difference higher than a factor of ten compared to the LC-HRMS/MS approach. This was most probably due to mixed spectra with matrix components resulting in not meeting the identification criteria. In conclusion, the obtained LOIs were acceptable for a broad urine screening. However, it should be kept in mind that LOIs of some particular compounds not studied here might also not meet required detectabilities. Therefore, LOIs of compounds of interest should be determined in addition to ensure their reliable detection. The possibility of a focused screening on targets of interest was additionally tested. Here, a PRM mode was added to the method, but with only a one-minute acquisition in the middle of the run for not losing the general screening ability for other compounds in contrast to classic target screening. This feature led to LOI improvements for 13 of 20 drugs with highest impact on that of bisoprolol. It represented a powerful tool to achieve higher sensitivity for selected analytes. However, the beneficial effect decreased with the number of targets within the inclusion list due to the one-minute scan time window (data not shown). Therefore, inclusion of not more than approximately 50 targets is recommended. If a higher target number is requested, either an apparatus with higher scan speed might be used or the scan window might be expanded, however with loss of sensitivity for other compounds. The possibility to reanalyze a liquid sample extract represents one advantage of non-dried samples. For PSI, however, there were no data published about the actual amount of eluted


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analytes from a sample. Thus, its reanalyzability was unknown and therefore addressed in this study. The relative signal in full scan (n = 3) of the tested drugs in eight runs is given in Table S1, supplied as Supporting Information. Samples where the corresponding drug could be identified are marked with a “*”. In the first reanalyzed samples (run 2), the relative signal ranged from 116-2% with 13 of 20 drugs showing values >50%. Up to run 4, signals of almost all drugs decreased to values ≤10% and up to run 6 to ≤1%. Higher values were only observed for diazepam and drugs with described carry-over (amphetamine, flufenamic acid, levetiracetam pentobarbital, and risperidone). For these drugs, identification was possible up to run 8. All other drugs could only be identified up to run 4. It seemed that the tendency for reanalyzable drugs decreased with their increasing lipophilicity, i.e. logP values31, which could be explained by their weak retention on the paper and consequently fast elution. In general, reanalyzing of spiked cartridges is not recommended, but might be possible once if e.g. no urine specimen was left for another workup. Application to Proficiency Test Samples. In the three proficiency tests for systematic toxicological analysis, all 23 spiked drugs could be detected. Unfortunately, the analyte concentrations were not given by the test provider. Proficiency test no. 1 consisted of amitriptyline, citalopram, doxepin, metoprolol, olanzapine, opipramol, paracetamol, and tramadol, test no. 2 of bisacodyl, clozapine, codeine, diazepam, doxylamine, ketamine, nordazepam, and olanzapine, and test no. 3 of bisacodyl, clozapine, 2-ethylidene-1,5-dimethyl3,3-diphenylpyrrolidine (EDDP, methadone metabolite), lamotrigine, metoprolol, olanzapine, and sertraline. Screening Results for the Authentic Human Urine Samples and Comparison with those of the LC-HR-MS/MS Approaches After UglucP or DUSglucE. In the previous study using


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LC-HR-MS/MS,29 a total number of 770 drug intakes (detected drugs; also by LC-MSn or GCMS screening) could be found in the 103 authentic urine samples with unknown concentrations, whereby 682 hits from these were detected after UglucP and 590 hits after DUSglucE. Using PSI-HR-MS/MS, in this study 565 drug hits could be identified, but among them seven additionally presumptive identified drugs raising the total drug intakes to 777. In particular three times agomelatine and once chlorphenamine, mitraciliatine, mitragynine, and promethazine, respectively. However, it should be mentioned again that the risk of false positive results is higher in PSI due to the lack of chromatographic separation and retention times. Nevertheless, there was no hint for that on these seven drugs. All identified drugs belonged to 50 different categories as divided by Maurer et al.,41 but were summarized into eight drug groups for better result description: anticonvulsants, antidepressants, benzodiazepines/Z-drugs, cardiovascular drugs, neuroleptics, opioids, stimulants/psychedelics, and others. In Table S-2, supplied as Supporting Information, drugs are given, which were found by the PSI-HR-MS/MS or by the LC-HR-MS/MS screening approaches after UglucP or DUSglucE. The number of drug intakes, categories, and drug groups are also listed. For comparison, the drugs detected by the particular screening approaches were calculated in relation to all drug intakes within particular drug groups. Results of these calculations are depicted in Figure 3. It should be mentioned again that the issue of analyte-carry over led to the need of conscientious screening inspection to avoid false positive results. Between 42-86% of the drug intakes were detected in the different groups by the PSI approach. The highest values were observed for anticonvulsants and stimulants/psychedelics and the lowest value for benzodiazepines/Z-drugs. It could be noticed that especially some hydrophilic and low mass drugs such as amphetamine, pregabalin, or valproic acid showed good


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results. In the group of benzodiazepines/Z-drugs, one reason for their low detected drug intake value might be low urine concentrations. At least for diazepam, corresponding urine concentrations should be below its LOI of 20 ng/mL and thus of minor relevance comparing them to the recommended cut-off level of 200 ng/mL for urine screening.40 In the screening approach comparison, the paper spray-based approach was able to detect more drugs in the groups of anticonvulsants (+11%: 75 vs. 86%) and stimulants/psychedelics (+4%: 81 vs. 86%) compared to the LC-HR-MS/MS approach after UglucP and more drugs in the groups of anticonvulsants (+25%: 61 vs. 86%), stimulants/psychedelics (+13%: 72 vs. 86%), and others (+1%: 73 vs. 74%) after DUSglucE. For all other groups, fewer drug intakes were observed by PSI-HR-MS/MS screening. However, except for benzodiazepines/Z-drugs, they showed at least similar value ranges between 68-86% compared to those by LC-HR-MS/MS approaches, which were between 75-93% after UglucP and 61-82% after DUSglucE. As an overview, the percentage of drugs detected by the three screening approaches in relation to all 777 drug intakes is given in Figure 4, a Venn diagram for an overview at a glance is depicted in Figure 5. The PSI-HR-MS/MS screening approach was able to detect 73% (565 hits) of all 777 drug intakes in the authentic human urine samples. The number of detected drugs was lower compared to those found by the LC-HR-MS/MS approaches after UglucP with 88% (682 hits) or after DUSglucE with 76% (590 hits). Despite different urine volumes of the approaches (18 µL urine finally in the system for PSI-HR-MS/MS, 20 µL for LC-HR-MS/MS after UglucP, and 10 µL after DUSglucE), the main reasons for discrepancies should be the higher matrix effects observed for PSI-HR-MS/MS with the consequence of higher LOIs for some drugs. Nevertheless, the PSI-HR-MS/MS results were acceptable for most screening purposes as the urine concentrations of the missed drugs might have been rather low as discussed above e.g. for


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diazepam. For analysis in context of e.g. intoxication investigations, drying time might be skippable with immediate sample analysis to further reduce turnaround time. However, the impact on ionization and elution properties should be investigated first. Comparing the new approach with the two conventional procedures, cleavage of conjugates was still necessary, but extraction and chromatographic separation of the analytes could be avoided. Overall, the PSI technology seems to be a promising alternative also for comprehensive urine screening of different drug classes.

CONCLUSIONS The presented PSI-HR-MS/MS screening approach was successfully evaluated. Despite the high matrix effects for most drugs, the obtained LOIs were acceptable for a broad urine screening and fulfilled international cut-off recommendations for the tested drugs of abuse. However, limitations should be considered such as detection of drugs in low concentrations and the risks of analyte carry-over or of false positive/negative results caused by mixed spectra. In addition, the described focused screening on targets of interest showed to be a powerful tool to achieve higher sensitivity for selected analytes without losing general screening ability. Stored paper spray cartridges could only be reanalyzed for particular drugs. The general screening proved its applicability by analyses of three proficiency tests for systematic toxicological analysis and of 103 authentic human urine samples. In the latter, the approach was able to detect 73% of all drug intakes, while LC-HR-MS/MS screening approaches after UglucP or DUSglucE detected slightly more drugs. Nevertheless, the PSI-HR-MS/MS results showed that this technology could become a promising alternative for comprehensive urine screening of different drug classes with the potential of reducing workload, analysis time as well as transport and storage costs in case of


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extending its use as sampling strategy. However, some problems should be solved before implementation in routine analysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at xxx Table S-1. Relative Signal in Full Scan (n = 3) of the Drugs Included in Mixture A in Eight Runs of Reanalyzed Sample Cartridges. Drugs Identified by MS/MS in the Particular Runs are Marked with a “*”. Table S-2. Drugs detected in the authentic human urine samples by the paper spray ionization (PSI)-HR-MS/MS screening approach or by the LC-HR-MS/MS screening approaches after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup (DUSglucE) as well as total drug intakes (positive urine samples for the given drug; detected also by LC-MSn or GC-MS screening), categories, and drug groups.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] *Fax: +49-6841-1626051 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources No funding


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors like to thank Achim T. Caspar, Sascha K. Manier, Lilian H. J. Richter, Lea Wagmann, Carsten Schröder, Gabriele Ulrich, and Armin A. Weber, for their support and/or helpful discussion and Thermo Fisher Scientific for providing a seed unit of the apparatus.


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(28) Helfer, A. G.; Michely, J. A.; Weber, A. A.; Meyer, M. R.; Maurer, H. H. J Chromatogr B Analyt Technol Biomed Life Sci 2017, 1043, 138-149. (29) Michely, J. A.; Meyer, M. R.; Maurer, H. H. Drug Test Anal 2017, DOI: 10.1002/dta.2255. (30) Maurer, H. H.; Meyer, M. R.; Helfer, A. G.; Weber, A. A. Maurer/Meyer/Helfer/Weber MMHW LC-HR-MS/MS library of drugs, poisons, and their metabolites; Wiley-VCH: Weinheim, Germany, 2017. (31) Michely, J. A.; Meyer, M. R.; Maurer, H. H. Anal Chim Acta 2017, 982, 112-121. (32) EMA. European Medicines Agency, Guideline on bioanalytical method validation; http://www.ema.europa.eu/ema/index.jsp?curl=pages/includes/document/document_detail.jsp?w ebContentId=WC500109686&mid=WC0b01ac058009a3dc, 2011. (33) Peters, F. T.; Drummer, O. H.; Musshoff, F. Forensic Sci Int 2007, 165, 216-224. (34) Remane, D.; Meyer, M. R.; Wissenbach, D. K.; Maurer, H. H. Rapid Commun. Mass Spectrom. 2010, 24, 3103-3108. (35) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 2003, 75, 30193030. (36) Vega, C.; Spence, C.; Zhang, C.; Bills, B. J.; Manicke, N. E. J. Am. Soc. Mass. Spectrom. 2016, 27, 726-734. (37) Wissenbach, D. K.; Meyer, M. R.; Remane, D.; Philipp, A. A.; Weber, A. A.; Maurer, H. H. Anal Bioanal Chem 2011, 400, 3481-3489. (38) Brown, D. H.; Hansson, R.; Oosthuizen, F.; Sumner, N. Drug Test Anal 2016, 8, 344-350. (39) Baselt, R. C. Disposition of toxic drugs and chemicals in man, 9th ed. ed.; Biomedical Publications: Seal Beach, CA, 2011. (40) Taskinen, S.; Beck, O.; Bosch, T.; Brcak, M.; Carmichael, D.; Fucci, N.; George, C.; Piper, M.; Salomone, A.; Schielen, W., et al. Drug Test Anal 2017, 9, 853-865. (41) Maurer, H. H.; Pfleger, K.; Weber, A. A. Mass spectral data of drugs, poisons, pesticides, pollutants and their metabolites; Wiley-VCH: Weinheim, 2016.


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Table 1. Ionization Effects by Co-eluting Analytes and Matrix Effect with Corresponding Coefficient of Variation (CV) drug

ionization effects by matrix effect (±CV), % co-eluting analytes (±CV), %

Amiodarone

-5 (±40)

-95 (±2)

Amitriptyline

-12 (±35)

-74 (±7)

Amphetamine

-26 (±24)

-91 (±8)

Bisoprolol

-8 (±38)

-77 (±6)

Clonidine

-21 (±12)

-70 (±6)

Codeine

0 (±6)

-91 (±2)

Diazepam

-8 (±28)

-88 (±3)

Diclofenac

-16 (±29)

-66 (±20)

Diphenhydramine -16 (±14)

-73 (±8)

Flufenamic acid

-19 (±21)

-18 (±30)

Levetiracetam

+23 (±41)

-98 (±11)

Losartan

+9 (±24)

-92 (±3)

Morphine

-21 (±20)

-81 (±4)

Pantoprazole

-20 (±25)

-93 (±2)

Pentobarbital

-2 (±41)

-44 (±28)

Promethazine

-26 (±18)

-75 (±7)

Quetiapine

-11 (±19)

-78 (±5)

Risperidone

-31 (±14)

-82 (±4)

Sertraline

+2 (±19)

-92 (±2)

Thiopental

-7 (±44)

-62 (±24)


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Table 2. Determined Limits of Identification (LOIs) of the Drugs Included in Mixture A by General and Focused Screening Using the PSI-HR-MS/MS Approach as well as Published LOIs Using a LC-HR-MS/MS Approach After Simple Urine Precipitation by Helfer et al.8

drug

PSI-HR-MS/MS PSI-HR-MS/MS LC-HR-MS/MS general screening focused screening general screening LOI, ng/mL LOI, ng/mL LOI, ng/mL

Amiodarone

400

200

-

Amitriptyline

40

20

10

Amphetamine

60

20

100

Bisoprolol

600

40

10

Clonidine

200

60

-

Codeine

200

200

100

Diazepam

20

20

10

Diclofenac

600

600

100

Diphenhydramine 10

10

10

Flufenamic acid

80

10

-

Levetiracetam

10,000

1,000

1,000

Losartan

20

20

100

Morphine

100

80

100

Pantoprazole

400

200

-

Pentobarbital

10,000

800

-

Promethazine

10

10

10

Quetiapine

10

10

10

Risperidone

100

10

10

Sertraline

600

400

100

Thiopental

10,000

800

-


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Legends to Figures

Figure 1. Reconstructed HR-MS ion chronograms of the exact protonated molecule masses (±5 ppm) of amiodarone, bisoprolol, morphine, and codeine-6-glucuronide with the corresponding logP value.31

Figure 2. HR-MS/MS reference spectra30 of morphine (A), an endogenous biomolecule (B), and their mixed spectrum in a spiked urine sample (1,000 ng/mL; C).

Figure 3. Drugs, divided into eight groups, detected by the paper spray ionization (PSI)-HRMS/MS screening approach or by the LC-HR-MS/MS screening approaches after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup (DUSglucE) in relation to all drug intakes of the particular groups. “n” indicates the number of drug intakes representing 100% for each group.

Figure 4. Percentage of drugs detected by the paper spray ionization (PSI)-HR-MS/MS screening or by the LC-HR-MS/MS screening after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup (DUSglucE) in relation to all 777 drug intakes.

Figure 5. Venn diagram describing the overlap of drug intakes detected by the paper spray ionization (PSI)-HR-MS/MS) and the LC-HR-MS/MS screening after urine precipitation with conjugate cleavage (UglucP) or after dried urine spot workup (DUSglucE).


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Figure 1. Reconstructed HR-MS ion chronograms of the exact protonated molecule masses (±5 ppm) of amiodarone, bisoprolol, morphine, and codeine-6-glucuronide with the corresponding logP value. 210x217mm (300 x 300 DPI)


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Figure 2. HR-MS/MS reference spectra of morphine (A), an endogenous biomolecule (B), and their mixed spectrum in a spiked urine sample (1,000 ng/mL; C). 165x136mm (300 x 300 DPI)



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Figure 3. Drugs, divided into eight groups, detected by the paper spray ionization (PSI)-HR-MS/MS screening approach or by the LC-HR-MS/MS screening approaches after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup (DUSglucE) in relation to all drug intakes of the particular groups. “n” indicates the number of drug intakes representing 100% for each group. 175x110mm (300 x 300 DPI)


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Figure 4. Percentage of drugs detected by the paper spray ionization (PSI)-HR-MS/MS screening or by the LC-HR-MS/MS screening after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup (DUSglucE) in relation to all 777 drug intakes. 175x331mm (300 x 300 DPI)



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Figure 5. Venn diagram describing the overlap of drug intakes detected by the paper spray ionization (PSI)HR-MS/MS) and the LC-HR-MS/MS screening after urine precipitation with conjugate cleavage (UglucP) or after dried urine spot workup (DUSglucE). 162x110mm (300 x 300 DPI)


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Paper Spray Ionization Coupled to High ... - ACS Publications

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