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

	Cathinone is a monoamine alkaloid found in the Khat plant (

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 These initial derivatives were designed for medical purposes; however, most were withdrawn as a result of abuse and obvious dependency among users.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

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

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

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

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

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

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

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

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

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

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


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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Articles

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

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

Novel ionization processes provide gas-phase ions of a wide variety of materials using mass spectrometry (MS). In the case of inlet ionization, the sub-atmospheric pressure region of the mass spectrometer becomes the "ion source." These simple and sensitive methods operate from solution or a solid matrix. Matrix-assisted ionization mass spectrometry (MAI-MS) uses a solid matrix spontaneously producing gas-phase analyte ions upon exposure to sub-atmospheric pressure without the need for any external energy input. The solvent-based method of solvent-assisted ionization mass spectrometry (SAI-MS) ionizes certain classes of compounds more efficiently than electrospray ionization mass spectrometry (ESI-MS), and voltage solvent-assisted ionization mass spectrometry (vSAI-MS) is a highly sensitive combination of ESI and SAI. Direct analysis of tissue, fluids, including whole blood and urine, biological extracts, reaction mixtures, and buffered, salty solutions detecting drugs, metabolites, lipids, and proteins, simultaneously, demonstrates the advantages of the multi-ionization concept for obtaining more comprehensive analyses from 384-well plates and, if desired, hyphenated with liquid chromatography (LC), further simplifying complexity. The methodology developed for inlet ionization allows rapid switching between MAI, SAI, vSAI, and conventional ESI increasing the comprehensiveness of mass measurements at equal or better mass and drift time resolution.

	Advancing chemical analysis through fundamental research and applying this knowledge to improve analyses in biological sciences will impact areas such as medical diagnostics (1). Mass spectrometry (MS), because of its specificity, sensitivity, dynamic range, wide applicability, and potential for high-throughput, is widely used in biological analyses. MS directly provides atomic and molecular composition information of heterogeneous bulk samples and surfaces, important for educated decisions on materials composition associated with quality, performance, safety, and more recently disease diagnostics. Integral to advances in MS are developments for transferring molecules into the gas phase regardless of size or volatility. As was demonstrated with electrospray ionization (ESI) and matrix-assisted laser desorption–ionization (MALDI), successful new ionization methods can have an impact on science far beyond anything envisioned in their early discovery, as witnessed by the award of a portion of the Nobel Prize in Chemistry to John Fenn and Koichi Tanaka in 2002. ESI and MALDI have strengths and weaknesses relative to analytical needs, which is the case with all ionization methods used with MS. Mass spectrometer manufacturers have recognized the need for multiple ionization methods by designing ion sources capable of ESI, atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) as well as offering AP-MALDI and intermediate pressure MALDI on some of these instruments. Ambient ionization methods such as atmospheric solids analysis probe (ASAP) (2), desorption electrospray ionization (DESI) (3), and direct analysis in real time (DART) (4) have also become popular. In this article, we describe new ionization techniques and manual and automated approaches that allow rapid switching between these ionization techniques as well as with ESI. The combination of methods allows interfacing with liquid separation methods, as well as direct analysis of samples, and increases the likelihood that important compounds will not be missed because of ion suppression or poor ionization efficiency.

	New means of transferring small, large, volatile, and nonvolatile compounds from the solid or liquid state directly into gas-phase ions were discovered through fundamental research and developed into methods providing exceptional sensitivity and simplicity (5). The initial discovery began using laser ablation of common MALDI matrices at AP, but instead of singly charged ions expected of MALDI, multiply charged ions expected of ESI were observed (6,7). Because the mass spectra looked like ESI but used laser ablation, the method was termed 

 (Figure 1a). Fundamental studies led to the unanticipated finding that the laser is unnecessary because analyte ions are formed when neutral matrix–analyte enters a heated inlet tube of a mass spectrometer (Figure 1b) (8,9). Further studies showed that the matrix could be the solvent used to dissolve the analyte (Figure 1c). Use of a solid matrix was termed

(11,12). While the method of introduction of the matrix sample into the heated inlet tube, which becomes the ion source, differs in LSI, MAI, and SAI, the results relative to charge states are the same, and thus the representative term 

 (13,14). However, the heated inlet tube was found not to be necessary provided that heat is applied through laser absorption by the matrix, or astonishingly, without any added external energy by using an appropriate volatile matrix (15,16). In all cases, multiply protonated analyte ions are formed from compounds having multiple basic sites, such as peptides and proteins, just as in ESI. Voltages, obstructions, or additives can improve ionization in these new ionization processes (9,16–21).


	Figure 1: Graphical representations of inlet ionization methods: (a) LSI in transmission geometry, (b) MAI vacuum cleaner, (c) SAI liquid junction pen, and (d) DAII (droplet assisted inlet ionization). 

	Drugs (verapamil, fexofenadine, erythromycin, azithromycin, and hydroxychloroquine), peptides (leucine-enkephalin, bradykinin, and angiotensin I), proteins (bovine insulin, ubiquitin, and cytochrome c), LB broth "Lennox", and 3-nitrobenzonitrile (3-NBN) were purchased from Sigma Aldrich. High performance liquid chromatography-grade water, methanol, acetonitrile, and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific Inc. and absolute ethanol from Decon laboratories. All chemicals were used without further purification.

	Stock solutions of drugs were prepared in ethanol (verapamil and azithromycin), 1:1 acetonitrile–water (erythromycin and hydroxychloroquine), and DMSO (fexofenadine). For peptides and small proteins, stock solutions of 1 mg/mL were prepared in water. Dilutions were made in 50:50 methanol–water (drugs) and in water (peptides and small proteins) to a final concentration of 5 µM, unless otherwise noted in the text. For MAI using the automation platform, protein samples were prepared in 1:1 

 methanol–water solutions and 100 mg of 3-NBN matrix was prepared in 3 mL of 3:1 

 acetonitrile–water solution. For the bacteria extraction protocol, 50 mL of bacterial strains was grown to stationary phase from overnight starter cultures at 37 °C and 200 rpm on LB media. The cultures were pelleted at 4 °C for 15 min at 4000× g and washed 3× times with cold water. The pellets were resuspended in double distilled (dd) water and lysed with an ultra-sonicator by alternating 10 s on/off cycle while on ice. Cell debris was separated by centrifugation at 16,000

, 4 °C for 15 min and 1.0 mL of the supernatant collected with two methanol–water extractions and dried on a biodryer, frozen, and lyophilized. The samples were then resuspended in ethanol. For the MALDI source, the matrix–analyte mixture was prepared in 1:3 

 and 1 µL of the mixture was spotted on the sample plate.

	Thermo Fisher Scientific Orbitrap Exactive and Waters Synapt G2S mass spectrometers were used. The Orbitrap Exactive was operated at 100,000 mass resolution (mass-to-charge [

] 200, 50%). The Synapt G2S instrument was operated in mobility-time-of-flight (TOF), sensitivity, and positive-ion mode detection. MAI-MS using the MSTM multifunctional automation platform was operated on the Synapt G2S mass spectrometer. The MSTM automation platform replaced the Waters ion source housing and was mounted on the instrument using a specially designed flange (22). For the automation measurements of proteins, 0.2 µL of 3-NBN and 0.1 µL of the protein solution was aspirated using a fused-silica capillary (internal diameter [i.d.] ~100 µm), and upon dispensing at the tip of the fused silica capillary allowed to dry for a few seconds prior to injection into a modified inlet tube for mass spectral analysis. For the bacterial strain, mass spectra were obtained using ESI by spraying 1.0 µL of the extract with 3 kV applied on the source, and MAI using 0.1 µL of the extract combined with 0.1 µL of the 3-NBN matrix solution, air dried and then transferred into the modified inlet. The source temperature was set at 80 °C. For MAI-MS using the MALDI source, no laser and low energy settings were used: sample plate 0–10 V, extraction 10 V, hexapole 10 V, and aperture "0" 5 V.

	For those mass spectrometers that provide a heated inlet tube, ionization is readily achieved for any of the inlet ionization methods after the source is removed for access to the inlet aperture of the mass spectrometer. Examples are shown in Figure 1, with and without the use of a laser, in which various surfaces were sampled using, for example, a pen through which fused-silica tubing was held in place to form a liquid junction (Figure 1c). The solution, after contacting a surface, was sucked into the section of fused silica tubing, the exit end of which was inside the heated inlet tube and, therefore, at sub-AP (22) similar to previous studies (23,24).

	Some of the small-molecule MAI matrix compounds, especially those which spontaneously produce analyte ions when exposed to sub-AP, have no labile hydrogen atoms and thus cannot directly donate protons (25). A common feature of all matrices which spontaneously produce analyte ions when subjected to sub-AP is that they sublime under the conditions of the experiment (26). Without sublimation, no ions are observed from these matrices. It is therefore surprising that such a fundamental chemical process as sublimation might still harbor secrets. Protic solvents, such as water or methanol, present in at least a few percent, enhance ionization in MAI, and especially of a nonvolatile analyte. However, drying the matrix sample prior to insertion into sub-AP does not hinder the ionization process, thereby demonstrating that ionization does not occur through liquid droplets. Studies to date suggest solid matrix particles are the engine of charge separation and the solvent the proton source. This means that ESI-like charge states are produced not only from charged liquid droplets as in ESI, but also from solid charged particles. On the other hand, SAI, in which the matrix is the solvent used to dissolve the analyte, produces ions most effectively in a heated inlet tube linking AP and the vacuum of the mass analyzer without application of a voltage, and must produce ions through highly charged liquid droplets similar to ESI. However, SAI and ESI ionize many compounds with different efficiencies. A hybrid method that combines the attributes of ESI and SAI, and termed 

, where a voltage is applied to a solution introduced directly into a heated inlet tube through fused-silica tubing, enhances sensitivity and combines attributes of ESI and SAI. The potential for high-throughput and automated analyses were previously demonstrated for MAI and SAI (27,28), and tissue imaging applications for LSI (29–31).

	The inlet ionization methods of MAI, SAI, and vSAI offer highly sensitive direct ionization methods using solid matrices like MALDI, but without a laser, a solution like ESI, but without the need of a voltage, and the solution method with a voltage that combines attributes of ESI and SAI. Each of these ionization methods offers compound-specific ionization efficiencies. For example, SAI has been shown to ionize certain steroids more efficiently than ESI (32) and MAI has low ionization efficiency for compounds that typically ionize by metal cation attachment and high efficiency for ionization of compound classes such as drugs and peptides. MAI, using 3-NBN as a matrix, ionizes typical background compounds with a low efficiency, which can be advantageous for trace analyses as was demonstrated for a sample of the peptide angiotensin I consuming just 5 femtomoles to produce a clean mass spectrum (33). This attribute makes MAI ideal for the analysis of urine, blood, and tissue samples for drugs and their metabolites, and proteins from buffered and detergent conditions, as demonstrated for the Ebola virus protein and bacteriorhodopsin, a membrane protein, as well as others (34–37). Metal cation adducts or other undesired attachments, for example matrix molecules, produced using MALDI or ESI are not observed with MAI matrices. Negative ions are spontaneously produced allowing, for example, detection of ganglioside and other lipids, such as cardiolipins, directly from a simple mitochondria extract, and carbohydrate conjugates as singly and multiply deprotonated ions with minimal fragmentation, which is often not the case using ESI or MALDI (38,39). Combining these new ionization methods with ESI in a single, easy-to-use platform gives the user the best chance to rapidly analyze compounds easily missed if only ESI, only MALDI, or only a single inlet ionization method are used for the analysis.

	With the manual platform, the liquid introduction methods of ESI, SAI, and vSAI can be interfaced with an liquid chromatography (LC) system for LC–MS analyses. Ion intensities, in an LC–MS study, using each of the liquid introduction methods with the drugs, erythromycin (MW 733) and hydroxychloroquine (MW 335), and the peptide, leucine-enkephalin (MW 555), were determined for injection of 1 µL of solution (Table I). The same ion intensity trends were observed at higher concentrations, vividly showing differences in ionization efficiencies between the ionization methods. Additional supporting information relative to ionization efficiencies of ESI, SAI, and vSAI using LC–MS has been provided in a previous publication (19).


	Figure 2: Comparison of MV1184 E. coli bacterial extract in 100% ethanol using the automated platform (22) and ionized by (a) ESI-MS and (b) MAI-MS on a Synapt G2S instrument.

	As might be expected, there are also differences in mass spectra obtained by direct ionization using MAI, MALDI, and ESI. The differences are best seen when comparing complex mixtures. One example is the comparison of ESI and MAI using the automated system which is capable of analyzing 384-well microtiter plates or surfaces, for example glass plates, using ESI, MAI, SAI, or vSAI, just as with the manual system. Three strains of 

 bacteria were grown, lysed, extracted in ethanol, and five replicates of each extract were acquired by MS using MAI, with the 3-NBN matrix in 3:1 acetonitrile–water, as well as with ESI directly from the ethanol extract. The results for the 

 in ethanol are shown in Figure 2. Some of the differences observed are ionization by Na

 adduction in ESI versus protonation in MAI, for example 

 726 and 704, while other ions, for example 

 637 in MAI, are not observed in both mass spectra. Because protonated ions fragment more readily, the MAI mass spectrum shows mass differences of 

 704 to 563), likely representing loss of the polar head group from a glycerophospholipid. Thus, some of the contents of this extract as viewed by these two ionization methods would seem to be different.


	Figure 3: (a) Total ion chromatogram of 50 acquisitions and (b) MAI mass spectra of protein solutions using 3-NBN: (1) bovine insulin (MW 5733), (2) cytochrome c (MW 12384), and (3) ubiquitin (MW 8560). The mass spectra were acquired using the automated platform (22) on a Synapt G2S instrument.

	An example of an automated acquisition of three proteins multiple times is shown in Figure 3 using MAI with 3-NBN as a matrix. Note that the mass spectra look nearly identical in charge states to ESI, but here ionization is from a solid rather than a liquid matrix. MAI has some distinct advantages relative to ESI. For example, simply using a different matrix provides different selectivity allowing ionization to be tailored to the problem, similar to MALDI. The most studied MAI matrices of the over 40 discovered so far are 3-NBN, 1,2-dicyanobenzene, 2-bromo-2-hydroxy-1,3-propandiol, and 2-methyl-2-hydroxy-1,3-propandiol, two of which have no labile hydrogen atoms, and yet all matrices produce singly or multiply protonated analyte ions similar to ESI. MAI, as noted earlier, does not require a heated inlet tube if the proper matrix is used. In fact, an intermediate pressure MALDI source can be used for analysis of multiple samples without the need for a laser (40,41). Figure 4 shows the analyses of six samples within 50 s in one row of a MALDI plate with samples spaced every other well. This approach has considerable potential if the time to introduce the plate into the ionization region can be greatly reduced from the current ca. 2 min. Interestingly, MAI has been reported to have equal or better mass and ion mobility resolution than ESI and MALDI on the same and other mass spectrometers (5,27).


	Figure 4: MAI fast analysis of six sample spots in 50 s: (a) Photograph of the sample plate with six spots (on each row) of the matrix–analyte mixture using 3-NBN as matrix, (b) total ion chromatogram, and (c) representative MAI mass spectra of the analytes from drugs (verapamil, MW 454; fexofenadine, MW 501; azithromycin, MW 748), peptides (bradykinin, MW 1059; angiotensin I, MW 1296), and small protein (bovine insulin, MW 5733). Data acquired using the commercial MALDI source of a Synapt G2S instrument.

	The SAI method of ionization also has advantages over conventional ionization for certain types of analyses. As with any new discoveries, the utility becomes most apparent when others begin to apply the new technology in innovative ways. For example, the Johnston group at the University of Delaware introduced droplet assisted inlet ionization (Figure 1d), which, for some compounds, provides up to four-orders of magnitude better ion sensitivity than ESI for direct aerosol analysis (42). Professor Eberlin at the University of Texas at Austin reported on an "MS pen" device for detection of cancerous tissue during surgery using solvents as the matrix in inlet ionization (43). There are a number of other examples where solvent matrices were used without application of high voltage as required with ESI, including zero volt paper spray (44–46). Imaging applications and MS/MS of multiply charged ions directly from surfaces have also been adapted by the group of Professor Li (University of Wisconsin) (47). MAI matrices have been used with ambient ionization sources with success (48,49). Professor Murray's group at Louisiana State University constructed a pulsed valve MAI device achieving 300 times more intense signal with 3-NBN than with 2,5-dihydroxyacetophenone, a common MALDI matrix (50). The width and breadth of use of the new ionization processes indicate flexibility and broadness of the initial inventions and findings.

	Fundamental studies relative to the new ionization technologies and methods for sample introduction continue to develop. The simplicity of ionization processes that need only exposure to the vacuum of the mass spectrometer or a heated inlet tube suggests applications where costs and simplicity are most critical. Developments in automated sample introduction with ever increasing speed and high sensitivity, and without carryover between samples or instrument contamination are being developed. Areas of application are anticipated to be in the general areas of clinical, pharmaceutical, and medical diagnostics applications, because of simplicity, speed, and robustness, but also field portable mass spectrometers because of the low pumping capacity requirements of MAI. The one thing that is clear is that the research so far has reshaped how one must think about ionization in a fundamental and applied fashion.

	NSF CHE-1411376 (to ST) and NSF STTR Phase II 1556043 (to CNM) are gratefully acknowledged. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation.

	(1) F.G. Strathmann and A.N. Hoofnagle, 

	(2) C.N. McEwen, R.G. McKay, and B.S. Larsen, 

	(3) Z. Takats, J.M. Wiseman, B. Gologan, and R.G. Cooks, 

	(4) R.B. Cody, J.A. Laramée, and H.D. Durst, 

	(6) S. Trimpin, E.D. Inutan, T.N. Herath, and C.N. McEwen, 

	(7) C.N. McEwen, B.S. Larsen, and S. Trimpin, 

	(8) C.N. McEwen, V. Pagnotti, E.D. Inutan, and S. Trimpin, 

	(9) J. Li, E.D. Inutan, B. Wang, C.B. Lietz, D.R. Green, C.D. Manly, A.L. Richards, D.D. Marshall, S. Lingenfelter, Y. Ren, and S. Trimpin, 

	(10) C.N. McEwen, V.S. Pagnotti, E.D. Inutan, and S. Trimpin, 

	(11) V.S. Pagnotti, N.D. Chubatyi, and C.N. McEwen, 

	(12) V.S. Pagnotti, S. Chakrabarty, B. Wang, S. Trimpin, and C.N. McEwen, 

	(13) C.B. Lietz, A.L. Richards, Y. Ren, and S. Trimpin, 

	(14) C.N. McEwen, V.S. Pagnotti, E.D. Inutan, and S. Trimpin, 

	(15) E.D. Inutan, B. Wang, and S. Trimpin, 

	(17) N.D. Chubatyi, T. Wang, and C.N. McEwen, 

	(18) V.S. Pagnotti, N.D. Chubatyi, A.F. Harron, and C.N. McEwen, 

	(19) M.A. Fenner, S. Chakrabarty, B. Wang, V.S. Pagnotti, K. Hoang, S. Trimpin, and C.N. McEwen, 

	(20) Z.J. Devereaux, C.A. Reynolds, J.L. Fischer, C.D. Foley, J.L. DeLeeuw, J. Wager-Miller, S.B. Narayan, K. Mackie, and S. Trimpin, S., 

	(22) S. Trimpin, S. Rauschenbach, K. Kern, I-C. Lu, E.D. Inutan, J.L. Fischer, S. Thawoos, Z. J. Devereaux, W.-J. Zhang, W. Zhang, M. Dillenburger, H.-J. Radar, K. Mullen, K. Hoang, A. Siderenko, M. Pophristic, and C.N. McEwen, "Fundamental Studies of Inlet Ionization with Sample Introduction at Atmospheric Pressure," paper presented at the 64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, Texas, 2016.

	(23) B. Wang, C.L. Dearring, J. Wager-Miller, K. Mackie, and S. Trimpin, 

	(24) S. Trimpin, B. Wang, C.B. Lietz, D.D. Marshall, A.L. Richards, and E.D. Inutan, 

	(26) S. Trimpin, C.A. Lutomski, T.J. El-Baba, D.W. Woodall, C.D. Foley, C.D. Manly, B. Wang, C.W. Liu, B.M. Harless, R. Kumar, L.F. Imperial, and E.D. Inutan, 

	(28) D.W. Woodall, B. Wang, E.D. Inutan, S.B. Narayan, and S. Trimpin, 

	(29) A.L. Richards, C.B. Lietz, J. Wager-Miller, K. Mackie, and S. Trimpin, 

	(30) E.D. Inutan, J. Wager-Miller, K. Mackie, and S. Trimpin, 

	(31) A.F. Harron, K. Hoang, and C.N. McEwen, 

	(32) N.D. Chubatyi, V.S. Pagnotti, C.M. Bentzley, and C.N. McEwen, 

	(33) K. Hoang, M. Pophristic, A.J. Horan, M.V. Johnston, and C.N. McEwen, 

	(34) E.D. Inutan, J. Wager-Miller, S.B. Narayan, K. Mackie, and S. Trimpin, 

	(35) D.D. Marshall, E.D. Inutan, B. Wang, C.W. Liu, S. Thawoos, J. Wager-Miller, K. Mackie, and S. Trimpin, 

	(36) S. Trimpin, S. Thawoos, C.D. Foley, D.W. Woodall, J. Li, E.D. Inutan, and P.M. Stemmer, 

	(37) S. Thawoos, J. Wager-Miller, E.D. Inutan, Z.J. Devereaux, C.D. Foley, Y.-H. Ahn, K. Mackie, P.M. Stemmer, and S. Trimpin, "Rapid Analysis of Proteins on High-Resolution Mass Spectrometers using Matrix-Assisted Ionization," paper presented at the 64th ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, Texas, 2016.

	(38) C. Reynolds, J. DeLeeuw, C. Corinne Lutomski, T. Sanderson, K. Przyklenk, and S. Trimpin, "Matrix-Assisted Ionization-Mass Spectrometry Enables Cardiolipin Characterization Directly From Intact Mitochondrial Membranes," paper presented at the 63rd ASMS Conference on Mass Spectrometry and Allied Topics, St. Louis, Missouri, 2015.

	(39) B. Wang, G. Liao, Z. Guo, and S. Trimpin, "Characterization of Carbohydrate-Monophosphoryl Lipid Conjugate Cancer Vaccine Candidates using Matrix Assisted Ionization Vacuum Mass Spectrometry," paper presented at the 62nd ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, Maryland, 2014.

	(42) A.J. Horan, M.J. Apsokardu, and M.V. Johnston, 

	(43) J. Zhang, J. Rector, J.Q. Lin, J.H. Young, M. Sans, N. Katta, N. Giese, W. Yu, C. Nagi, J. Suliburk, J. Liu, A. Bensussan, R.J. DeHoog, K.Y. Garza, B. Ludolph, A.G. Sorace, A. Syed, A. Zahedivash, T.E. Milner, and L.S. Eberlin, 

	(45) P. Wei, S. Bag, C.J. Pulliam, D.T. Snyder, and R.G. Cooks, 

	(46) M. Wleklinski, Y. Li, S. Bag, D. Sarkar, R. Narayanan, T. Pradeep, and R.G. Cooks, 

	(49) J.M. Santos, P.H. Vendramini, N.V. Schwab, M.H. Eberlin, and D.R. Morais, 

 is with the Department of Chemistry at Wayne State University in Detroit, Michigcan, and the Cardiovascular Research Institute at Wayne State University School of Medicine. 

Santosh Karki, Darrell D. Marshall, Ellen. D. Inutan and Anil K. Meher

 are with the Department of Chemistry at Wayne State University. 

Sara Madarshahian, Madeline A. Fenner and Charles N. McEwen

 are with the Department of Chemistry & Biochemistry at the University of the Sciences in Philadelphia, Pennsylvania. Direct correspondence to: 

Novel ionization processes provide gas-phase ions of a wide variety of materials using MS. These simple and sensitive methods operate from solution or a solid matrix. Both manual and automated platforms are described that allow rapid switching between the ionization methods of MAI, SAI, vSAI, and conventional ESI.

Combining Novel and Traditional Ionization Methods for Mass Spectrometry for More Comprehensive Analyses

Characterization of protein modifications is an essential aspect of biopharmaceutical development. Traditionally, the characterization process of chromatographic peaks involves manual, larger-scale fractionation to obtain a sufficient amount of material for further analytical studies. This article presents a fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization. This innovative technique significantly accelerates MS peak characterization compared to traditional approaches and avoids the risk of unintended modifications of the variants as a result of the isolation process, for example, deamidation during storage of isoforms. This approach considerably reduces the required sample amount and can be used for the characterization of product-related impurities during early stage development.

	Monoclonal antibodies (mAbs) are highly complex recombinant proteins used successfully as therapeutics in many diseases. These immunoglobulins are large tetrameric glycoproteins with a molecular weight of 150 kDa. Monoclonal antibodies for therapeutic use are produced in genetically modified mammalian cells, for example, Chinese hamster ovary cells. During the expression, process- and product-related side products, such as glycosylation variants, glycation, and mispairing for CrossMabs, are generated. Additional modifications can occur during downstream processing and storage, including deamidation, isomerization, oxidation, and truncation (1–3). Aspartic acid or asparagine residues in the light and heavy chain are known to form succinimide under mildly acidic conditions. Succinimide further isomerizes to isoaspartic acid or back to aspartic acid under neutral or alkaline conditions. Hotspots for aspartic acid isomerization and asparagine deamidation are the motifs DG, NT, and NG (4–7).

	These structural alterations or modifications lead to charge and size heterogeneity. For example, high-molecular-weight species, such as aggregates of mAbs, have been correlated to a high safety risk because of their immunogenicity and influence on pharmacokinetics and efficacy (8). The formation of isoaspartic acid from a succinimide reaction may lead to a loss of biological activity (6,9). Most of these modifications can be monitored by the commonly used separation techniques, ion-exchange chromatography (IEC) or size-exclusion chromatography (SEC), and, consequently, these methods are commonly used in biopharmaceutical quality control (QC) strategies.

	Characterization of charge and size variants is required to generate the product knowledge that forms the basis for critical quality attribute (CQA) assessment. The former is commonly performed using offline fractionation followed by mass spectrometry (MS) analysis of reduced protein in combination with enzymatic peptide maps. Although MS-friendly mobile phases for SEC separations have been reported recently (10,11), most buffers used in an IEC or SEC system are not compatible with MS. Therefore, an offline fractionation with subsequent up-concentration and buffer exchange step has to be performed. This procedure is very time-consuming and requires considerable amounts of isolated material (12), thereby often prohibiting peak characterization during early development. During the preparation, the isolated fraction is exposed to stress, which adds the risk of unintended modifications from sample preparation. To accurately determine the identification of the samples, further processing is performed including reduction, alkylation, and digestion steps (12–14).

	During conventional characterization of mAbs, whole antibody samples were reduced and digested by dithiotreitol (DTT) or immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS). The fragments were then separated by IEC and reversed phase in the two-dimensional liquid chromatography (2D-LC) setup using comprehensive mode (15,16). By using this approach, variants of a mAb sample can be characterized but not assigned to specific peaks. The scope of this work was the direct characterization of mAb variant peaks observed in IEC or SEC QC) routine methods in a fully automated workflow. Several attempts were made to establish semi- or full automation for this workflow using fraction samplers or custom-made systems (17,18). However, most of those solutions were either not fully automated or very complex to handle and set up (17). Tran 

 performed all steps of a peak characterization including an IEC separation, online reduction, and online digestion with subsequent mass analysis via electrospray ionization time-of-flight (ESI-TOF) for smaller proteins with a customized capillary chromatographic system using a stop-flow technique. However, a fully automated procedure allowing use of routine analytical methods common for quality control strategies has been lacking (18).

	This article describes a fully automated online reduction 2D-HPLC quadrupole time-of-flight (QTOF)-MS setup that allows rapid characterization of protein modifications with a multiple heart-cut valve system for automated peak fractionation. In contrast to earlier work, our process provides not only online reduction of trapped peaks but also a reversed-phase separation of the reduced proteins before entering the MS system. The developed system uses standard HPLC components and allows the use of the established QC methods for larger proteins (mAbs) in the first dimension without any adaptations. Peaks of interest observed in routine QC measurements can be directly characterized without the need for re-conformation runs. The use of a multiple heart-cut module allows for seamless analysis in the second dimension without the need to stop the flow. The system is easy to program with commercially available software. This article also discusses the benefits of an online approach versus standard offline peak characterization in detail.

-morpholino) ethanesulfonic acid monohydrate (MES), MES sodium salt, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dithiotreitol (DTT), sodium hydroxide (NaOH), and hydrochloric acid (HCL) were purchased from Merck KGaA. A 50% formic acid (FA) solution and phosphoric acid were obtained from Fluka (Sigma-Aldrich Chemie GmbH). Tris(hydroxymethyl)aminomethane (TRIS), piperazine, disodium hydrogen phosphate, sodium dihydrogen phosphate, 

(2-carboxyethyl)phosphine hydrochloride (TCEP), and sodium chloride were purchased from Sigma-Aldrich. Isopropyl alcohol (IPA) was acquired from Sigma-Aldrich. Potassium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate were ordered from Acros Organics. Purified water was used from a Milli-Q water Advantage A1-system (Merck KGaA).

	Experiments for online reduction of mAbs were performed on an Agilent 1290 Infinity 2D–LC system. The standard system itself consists of the following modules: 1260 quaternary pump (G5611A), 1290 binary pump (G4220A), 1260 Bio ALS autosampler (G5667A), 1290 thermostat (G1330B), 1290 column oven (G1316C) containing a six-port, two-position valve (G4231B), and two 1260 ultraviolet (UV) detectors (G1314F). The 2D interface module consists of a 1290 eight-port, two-position valve (G4236A) and two valve decks for multi heart-cut sampling with 6 × 40 µL loops each (G4242A multiple heart-cutting kit). To this standard system a third binary pump (G4220A) and a second column oven (G1316A) containing a six-port, two-position valve (G4231B) was added to enable the online reduction of antibodies. For MS detection, a Bruker Impact II ESI QTOF system (0211 G203) was used. The 2D-LC system was connected via a fused silica capillary with a diameter of 75 µm to the MS system. OpenLab CDS Chemstation (Agilent) was used to control the 2D-LC system. HyStar Software (Bruker) was used to control and acquire data from the MS system. A start signal for each individual 2D run was triggered by a customized software macro by means of a contact closure board. The signal of the 2D UV detector was fed into the Bruker software by an analog-to-digital (A/D) interface net ADC LC 421n (SCPA GmbH).

	Starting with injection of the sample and separation on the first column, peaks of interest were fractionated online and stored in one of the 40-µL loops. During fractionation of the first dimension one peak after the other was injected onto the second dimension where it was either reduced and separated, or separated and then analyzed in the MS system. The process from injection of the sample until analysis in the MS system was fully automated by a "Valve Event Plugin" Macro from ANGI (Gesellschaft für angewandte Informatik). Each time a fraction was injected into the second dimension the third pump was started again and a start signal was sent to the MS system. In this way, a separate MS data file is generated for each fraction. Another advantage of the macro is that a valve in the column oven can be switched time dependent so that no reducing agent or salt from the first dimension IEC or SEC enters the MS system. In the setup for online reduction the second dimension UV detector was excluded to get sharper peaks and a good separation in the MS total ion chromatogram (TIC).

	Three different mAbs and one antigen-binding fragment (Fab) were used as a sample for online reduction. For online reduction, the mAb can be injected directly without any dilution or preparation.

	IEC of mAbs was performed according to conditions listed in Table I. The columns used were from Thermo Scientific.

The cut fractions were directed to a 2.1 × 50 mm, 1.7-µm Acquity BEH C4 reversed phase column (Waters Corporation) and trapped. Mobile phase A was 0.1% FA in purified water (PWA) and mobile phase B was 0.1% FA in acetonitrile. The pump was set to deliver 99% mobile phase A and 1% mobile phase B at a flow rate of 0.5 mL/min. The column temperature was set to 70 °C.

	The loaded reversed phase trapping column was flushed with the reduction solution at a flow rate of 56 µL/min for 5 min. The reduction solution was prepared by either dissolving 0.1543 mg DTT or 0.2502 mg TCEP in 50 mL tris solution pH 7.5 ending up in a final concentration of 0.02 M DTT or TCEP. The reduction solutions were freshly prepared each day.

	The trapped and reduced peaks were finally separated by applying the gradient conditions as described in Table II.

	The complete characterization of a peak of interest consists of four steps (see Figure 1). After cutting the first peak of interest in Deck A, subsequent peaks were sampled in Deck B. Immediately after cutting, peak 1 was flushed to the reversed phase column where it is trapped and desalted. By switching valve 2, the trapped peak was reduced by pumping reducing reagent via the 3D pump. After reduction, valve 2 and valve 3 were switched simultaneously and the reduced mAb chains were separated with a reversed phase gradient and detected by MS.


	Figure 1: Flowchart online reduction. (a) 1D: Cutting of Peak 1 in Deck A, 2D: idle, 3D: re-equilibration of reversed-phase column. (b) 1D: Cutting of further peaks in Deck B, 2D: Peak 1 trapped on reversed-phase column, 3D: idle. (c) 1D: Cutting of further peaks in Deck B, 2D: idle, 3D: reduction of Peak 1. (d) 1D: Cutting of further peaks in Deck B, 2D: gradient separation of reduced Peak 1, 3D: idle.

	For detection of reduced chains as well as material that has not been reduced, a Bruker Impact II system was used. The detection range was set between 300 and 2500 

. The end plate offset was 500 volt and dry temperature was set to 220 °C. Calibration was performed with 0.005% phosphoric acid.

Development of an On-Column Reduction Step

	Complete on-column reduction of mAb samples was achieved after a 5-min flush with either 0.02 M DTT or 0.02 M TCEP (data not shown). Longer reduction times were also evaluated without any improvement. Based on this observation no further optimization was deemed necessary. To verify the designed setup with the 5-min flow through of the reducing agent, a CrossMab (19) antibody (Antibody A) with one extended Fab arm on one side (2+1 CrossMab [20]) was used. For IEC separation conditions refer to Table I. The size of the whole antibody is approximately 194 kDa without accounting for additional glycosylation. This antibody is built of two different heavy chains (HC) with a mass of approximately 77 kDa and 51 kDa and two different light chains (LC) with 22 kDa and 24 kDa; the chain with 24 kDa is present two times for each antibody. Figure 2a shows a TIC of the antibodies main peak (0.38 µg of protein for each fraction) after reduction. Major peaks could be assigned to the respective HC and LC chains.


	Figure 2: (a) Total ion chromatogram of the reduced antibody A: The peaks marked with a black arrow show the two light chains. Peaks with orange arrows represent the two different heavy chains of the antibody. Mass analysis of antibody A light chain. (b) and (c) Deconvoluted spectra of the two different light chains of the antibody, with molecular weight 21,957 Da and 23,617 Da. (d) and (e) Deconvoluted spectra of the two different heavy chains of the antibody, with molecular weight 50,635 Da and 77,247 Da.

	Figure 2b and 2c depict the deconvoluted masses of all heavy and light chain fractions after online reduction. Other peak spectra showed comparable spectra.

	Four different chains of the antibody A were detected after online reduction with excellent signal intensity (Figure 2). All interchain disulfide bridges as well as all intrachain disulfide bridges were reduced. The Fc glycosylation of the heavy chains was detected with different glycoforms. The detected masses corresponded to the theoretical masses based on the amino acid sequence of the four different chains (Figure 2). In addition, the intact antibody A (without reduction) was analyzed using 2D-LC–MS and the theoretical mass was observed (data not shown).

	The developed system was used to analyze unknown peaks in stressed samples of three different antibodies and one Fab Fragment (Fab 1). Antibody B is a standard IgG1 antibody (Trastuzumab), whereas antibody A and C have extended Fab arms on one side and therefore a higher molecular weight than typical IgGs.

Characterization of a Low Abundance Basic IEC Peak in Antibody B

	The IEC chromatogram of antibody B shows a low abundance peak in the basic region (Figure 3a). For IEC separation conditions refer to Table I. A 40-µL sample of the peak of interest was extracted (0.24 µg of protein for each fraction) in the first dimension and transferred to the second dimension reversed phase column for on-column protein reduction. The reduced species were then eluted using an acetonitrile gradient and analyzed by QTOF-MS. The obtained deconvoluted mass spectrum is presented in Figure 3b and 3c.


	Figure 3: (a) IEC chromatogram of first dimension separation of antibody B. The unknown peak is marked with a black arrow. (b) and (c) Deconvoluted MS spectrum antibody B, only the heavy chain is shown. (b) Reduced main peak as a reference. (c) MS spectrum of the heavy chain of the fractionated and online reduced basic peak. The succinimide versions are marked with an asterisk.

	The peak with a mass of 50,601 represents the G0 glycovariant, whereas 50,763 and 50,926 are the G1 and G2 variants (7) of the non-modified heavy chain. The deconvoluted spectrum of the basic peak displays significantly increased signals with a difference of 17 ± 1 Da for all three glycoforms. This difference, combined with the retention time shift to the basic region of the IEC, suggests isomerization of aspartic acid to succinimide in the heavy chain of the mAb. This conclusion is further supported by data already published (21). The already present shoulders in the main peak spectrum at the same position are method-induced artifacts and represent the loss of water. The same effect can be seen in the spectrum of the basic peak.

Characterization of a Low Abundance Acidic IEC Peak in Antibody C

	In a second experiment an acidic peak of an IEC separation of antibody C was analyzed (see Figure 4a). For separation conditions refer to Table I. A 40-µL fraction of the peak of interest (0.54 µg of protein for each fraction) was sampled, reduced, and analyzed by QTOF-MS. The main peak was analyzed as a reference. The results are presented in Figure 4b and 4c.


	Figure 4: (a) IEC chromatogram of first dimension separation of antibody C. The unknown peak is marked with a black arrow. (b) and (c) Deconvoluted MS spectrum antibody C, only the heavy chain is shown. (a) Reduced main peak with the expected distribution of the glyco variants G0, G1, and G2. (b) Reduced acid peak.

	The main peak shows a normal distribution of the glyco variants, with the most abundant variant being G0, followed by G1 and G2 (Figure 4b). In the spectrum of the analyzed acidic IEC peak (Figure 4c), G0 was dramatically reduced, while the G2 peak was significantly increased. This can be explained by glycation of the heavy chain. Glycation is the result of covalent linkage of glucose to a lysine via formation of a Schiff base (22). This leads to loss of a positive charge of the molecule and a shift to the acidic region of the IEC chromatogram. Glycation typically occurs during the CHO cell culture process of antibody production when a high level of glucose is available in the cell culture medium. Glycation of the G0 variant results in an isoform with the same mass as the G1 variant. In addition, the G1 variant is also glycated leading to the observed increased mass of 49,969 Da. Glycation of the G2 variant leads to a new peak with a mass of 50,131 Da, which is only observed in the isolated acidic peak and not in the MS spectrum of the IEC main peak. The elution behaviour of the IEC peak supports the hypothesized glycation.

Characterization of a Low Abundance Acidic IEC Peak in Fab 1

	In addition to monoclonal antibodies, a therapeutic Fab (Fab 1) was also characterized by 2D-HPLC–MS. The material was temperature stressed for 8 weeks at 40 °C and degradation products were subsequently measured by IEC. The non-stressed material was measured as reference. For separation conditions refer to Table I. The IEC chromatogram of the stressed material showed one unknown peak, which was subsequently analyzed with the established 2D-HPLC–MS online reduction assay. The peak of interest is shown in Figure 5a.


	Figure 5: (a) IEC chromatogram of first dimension separation of Fab 1 highlighting the peak of interest. (b) and (c) Deconvoluted mass spectra, only the light chain is shown: (b) Reduced main peak as a reference and (c) fractioned and online reduced acidic peak of the stressed Fab 1 material.

	The peak was analyzed and found to be a modified light chain with a mass delta of +16 Da in comparison to the non-modified light chain. Based on three-dimensional X-ray structure measurements (data not shown), this mass shift most probably represents an oxidation of an exposed tryptophan residue in the complementarity-determining region (CDR) of Fab 1.

	The deconvoluted MS spectra are depicted in Figure 5b and 5c. Because the fractionated peak is next to the main peak there is also a small signal of the native light chain, with the mass of 24,004 Da next to the modified version with a mass of 24,020 Da.

	Further experiments using SEC in the first dimension instead of IEC were performed. These tests were performed with antibody A and antibody B. The online trapping and reduction of fractionated peaks were comparable to the IEC experiments (data not shown here). This combination is most appropriate when mispaired species or fragment peaks have to be characterized.

	An alternative reducing agent, namely TCEP instead of DTT, was evaluated using exactly the same conditions such as temperature, concentration of the reducing agent, duration of reducing agent flow, and flow rate. TCEP was also tested with different antibodies and produced exactly the same results as the reduction with DTT (data not shown).

	Characterization of product variants or post-translational modifications of therapeutic antibodies is essential because of their potential impact on product quality, safety, and efficacy (23); for example, a loss in potency can become a problem (24,25). To control those species various analytical QC methods are applied that are predominantly HPLC-based. Until now the characterization of unknown peaks had to be performed by offline fractionation. This is a time-consuming process with the risk of introducing additional, unintended modifications. The 2D-HPLC–QTOF-MS setup described here enables fully automated and rapid characterization of unknown peaks and is easy to use. It was possible to increase the sensitivity fivefold (0.2 µg of protein for each cut) compared to previous works (17). The system uses standard HPLC components and allows the use of the established QC methods for larger proteins (such as mAbs) in the first dimension without any adaptations. Unknown peaks observed in routine QC measurements can be directly characterized without the need of re-conformation runs or bridging between methods. The use of multiple heart-cutting as part of the 2D-HPLC system offered easy analysis in the second dimension without the need to stop the chromatographic flow. This approach has been successfully implemented for a plethora of biopharmaceutical development projects in our pipeline. The use of a reversed phase gradient for the separation of reduced mAb chains enables the identification and localization of modifications. Compared to conventional peak fractionation and characterization, the 2D-HPLC–MS setup described here yields a reduction in time-to-result of one order of magnitude, allowing sample characterization in a single day (Figure 6).


	Figure 6: (a) Workflow for manual peak characterization. (b) Flow chart of an online peak characterization. Different peaks of interest from a LC (IEC or SEC) chromatogram are fractionated and directly analyzed reduced or none reduced in the MS system.

	In some cases (for example, deamidation) identification of the modification can be improved by further reducing the size of antibody chains by digestion enzymes, like papain or trypsin. Further development work is ongoing to establish an additional digestion step for each fractionated peak in a 4D-HPLC–MS setup to achieve clear localization of modifications and verification of small mass shifts (1 Da for deamidation).

	The work presented here was sponsored by the Roche Global Strategy Team (GST) process. The authors wish to thank Rico Schaerer from Agilent for his support.

	(1) K. Sandra, I. Vandenheede, and P. Sandra, 

	(7) C.A. Sha Sha, K. Brorson, D.-Y. Lee, and S. Yoon, 

	(9) A.C.B. Mire-Sluis, R. Madsen, A. Polozova, A. Rosenberg, H. Smith, T. Arora, and L. Narhi, 

	(19) C. Klein, W. Schaefer, and J.T. Regula, 

	(22) S. Fischer, J. Hoernschemeyer, and H.C. Mahler, 

European Journal of Pharmaceutics and Biopharmaceutics

J. Chromatogr. B Analyt. Technol. Biomed. 

 studied biochemistry at the Ruhr University Bochum, the Max-Planck Institute for Molecular Physiology, Dortmund, Germany, and the Scripps Research Institute, San Diego, California, USA. He went on to do his Ph.D. at the University of Oxford, UK, focusing on structural biology. Since 2011, Christian has worked for Roche and is currently focused on analytical development and quality control of protein therapeutics in a clinical setting. 

 is Group Head of a mass spectrometry laboratory at Hoffmann-La Roche, Biotech Analytics in Basel, Switzerland. She is an expert in MS protein characterization and hyphenated techniques. Anja studied biotechnology at the Fachhochschule Lausitz - University of Applied Sciences, Germany. 

 has worked as a principal scientist in the Biotech Analytics department of Hoffmann-La Roche since 2004. He studied analytical chemistry at the Reutlingen University, Germany. He has been involved in HPLC method development and validation for small and large molecules for his whole career. 

 is a PhD student in the Center for Proteomics and Metabolomics at the Leiden University Medical Center (LUMC), Leiden, in The Netherlands. Before that he studied biology at Regensburg University, Germany. 

 studied applied chemistry at Reutlingen University, Germany, with special focus on bioanalytics. Since 2017 Denis has worked at Hoffmann-La Roche as a senior associate in the Biotech Analytics department. Both Denis and Christoph worked on the development of the described characterization system during their master theses.

A fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent QTOF-MS characterization is presented. The technique is based on state-of-the-art 2D-HPLC technology coupled with additional HPLC modules via a dedicated software macro.

Rapid Online Reduction and Characterization of Protein Modifications Using Fully Automated Two-Dimensional High Performance Liquid Chromatography–Mass Spectrometry

A simple, high throughput sample preparation method has been developed and validated for quantitation of over 40 drugs of abuse and metabolites using a polymeric SPE plate. Urine samples were hydrolyzed with E. Coli recombinant β-glucuronidase and then extracted with a Panthera Deluxe SPE plate. The eluates were separated using a Gazelle C18 UHPLC column and detected using an ExionLC-API4500 mass spectrometer. For most drugs, both the extraction and hydrolysis recoveries were over 90%.

	In 2014, nearly two million Americans either abused or were dependent on prescription opioid pain relievers. Ninety one Americans die every day from an overdose involving either prescription opioids or heroin. Today's abuse profile combines both clinical and street drugs.

	Drugs of abuse are commonly tested in clinical labs using the dilute and shoot approach, which comes with high matrix interference at low concentration and long-term deterioration of the LC–MS system. The purpose of this study was to evaluate the solid phase extraction approach as an alternative. Combining efficient hydrolysis based on 

 recombinant β-glucuronidase with an automated extraction method enables robust and dependable results.

	All drugs of abuse standards, their isotope internal standards (IS), codeine-6-β-D-glucuronidase, and morphine-3-β-D-glucuronidase were purchased from Cerilliant (MA, USA). BG100 β-glucuronidase was provided by Biotec-La Piedra Biotechnologia SpA (Chile). 

 β-glucuronidase (BG) was purchased from Campell Science (IL, USA). The Panthera Deluxe Polymeric SPE plate (30 mg/well), the Celerity Deluxe Polymeric SPE plate (30 mg/well), the Orpheus C18 SPE plate (50 mg/well), and the Gazelle C18 2.1 mm I.D. × 50 mm UHPLC column (1.7 µm) were manufactured by Orochem Technologies Inc. (IL, USA). The two polymeric SPE products A and B were manufactured by competitors.

	The ExionLC-API4500 Triple Quad MS (AB Sciex) was operated in negative ion mode for barbiturates and THCA and in positive ion mode for the rest of the drugs.

	Orochem's EZYPRESS HT96-well plate positive pressure manifold unit was used for the SPE procedures. Evaporation was carried out using a Quikvap 96-well plate evaporator (Orochem Technologies Inc.).

	Human urine samples were fortified with standards at different concentrations. For the enzyme hydrolysis recovery test, human urine samples were fortified with codeine-6-β-D-glucuronide, morphine-3-β-D-glucuronide, and 6-MAM at ULOQ level (equal to 2000 ng/mL free drug level) or with THCA-glucuronide at HQC level (equal to 200 ng/mL free drug).

	Optimized SPE method for barbiturates and THCA: 0.1 mL of fortified urine was mixed with 0.05 mL of internal standard solution (IS) in 90% methanol solution, and 0.1 mL of pre-made mixture of 200 mM ammonium acetate pH 6.8 buffer and BG solution in 5/2 v/v ratio and incubated at 55 °C for 30 min. A Panthera Deluxe SPE plate (30 mg/well, 2 cc/well plate) was pre-conditioned with 1 mL of methanol followed by 1 mL of water. Hydrolyzed urine solution was loaded onto the extraction plate, which was then washed with 1 mL each of water and 20% methanol. The analytes were eluted with 1.5 mL of methanol. Solvent was evaporated under nitrogen at 45 °C and the analytes were then reconstituted with 0.5 mL of 30% methanol and injected into the LC–MS/MS.

	SPE method for other drugs and metabolites: A mixture of 0.25 mL of master mix (ammonium acetate buffer, 25 µL of BG100 glucuronidase and IS) and 0.1 mL of fortified urine was incubated at 68 °C for 30 min. A Panthera Deluxe SPE plate (30 mg/well, 2 cc/well plate) was pre-conditioned with 1 mL of methanol and then 1 mL of water. Hydrolyzed urine solution was loaded onto the extraction plate and then washed with 1 mL each of water and 5% methanol. The analytes were eluted with 1.0 mL of methanol and evaporated under nitrogen at 40 °C and then reconstituted with 0.3 mL of 5% methanol and injected into the LC–MS/MS.

	A Gazelle C18 (1.7 µm), 2.1 mm I.D. × 50 mm long UHPLC column was used for the separation. For the barbiturates and THCA, the gradient mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The gradient ran from 30% to 95% B. The rest of the compounds used a gradient mobile phase of 0.1% formic acid in water containing 20 mM of an ammonium buffer (A) and 0.1% formic acid in methanol (B). The gradient ran from 5% to 95% B.

	An ExionLC-API4500 Triple Quad MS (AB Sciex) was operated in negative ion mode for barbiturates and THCA and in positive ion mode for rest of the drugs.

	We have established that the Panthera Deluxe SPE plate gives better reproducibility and recovery than the Celerity Deluxe Plate, especially for THCA (Table I). It also shows better performance than the two competitor polymeric SPE products (Table II). The C18 plate gives poor recoveries for most compounds. The extraction recoveries were in the range of 74.2–116.9% for all drugs (Table III).

	Under current hydrolysis conditions, recoveries of codeine-6-β-glucuronide and morphine-3-β-glucuronide were 89.9% and 110.5%, respectively, while the recovery of labile analyte 6-MAM was 100.8%. Recovery of THCA-glucuronide was 89.5%.

	Compared to dilute and shoot (D&S), the Panthera Deluxe procedure gave overall better results demonstrating greater AMR and lower LLOQ (Table IV). Results for xenobiotic interference/effect testing and matrix induced ion suppression/enhancement were comparable between procedures, while Panthera SPE showed superior results in terms of matrix interference/effect tested at cut-off concentration. Response factors for barbiturates and THCA clearly improved with SPE (Figure 1).


	Figure 1: Mass chromatograms of fortified urine (at cut-off) processed by dilute and shoot (left) and the Panthera SPE process (right). Peaks in eluting order: phenobarbital, butabarbital, butalbital, amobarbital, secobarbital, and THCA.

	Several different mobile phases were tested. For most of the compounds, there was no significant difference in peak shapes with or without ammonium buffer; however, ammonium buffer did significantly improve benzodiazine's peak shapes. There was no significant fronting or tailing of the opiates using the Gazelle C18 UHPLC column (Figure 2).


	Figure 2: Mass chromatograms of morphine, oxymorphone, hydromorphone, codeine, oxycodone, noroxycodone, hydrocodone, norhydrocodone, and 6-MAM (peak from left to right) in fortified urine sample using the Panthera Deluxe SPE process.

	Orochem has developed fast and reliable SPE methods for the analysis of over 40 drugs of abuse in a urine toxicology setting. These improved methods demonstrate reduced matrix effects and expand the lower end of the AMR by almost an order of magnitude. The sample processing time per plate is about 15 min and the total cost of sample preparation is maintained at a very competitive level. Furthermore, Panthera SPE demonstrates improved performance over its predecessor, Celerity, and over two other major competitor products.


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High Throughput Sample Preparation Method for Drugs of Abuse in Urine


Orochem Technologies Inc.

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Special Issues-03-02-2018 | LCGC Supplements

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

Combining Novel and Traditional Ionization Methods for Mass Spectrometry for More Comprehensive Analyses

Rapid Online Reduction and Characterization of Protein Modifications Using Fully Automated Two-Dimensional High Performance Liquid Chromatography–Mass Spectrometry

Orochem Technologies Inc.

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