US 2015 0346170A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0346170 A1 Huang et al. (43) Pub. Date: Dec. 3, 2015

(54) EXTRACTION, DERIVATIZATION, AND Publication Classification QUANTIFICATION OF ANALYTES (51) Int. C. (71) Applicant: QUANTALYTICAL LABS INC., GOIN30/72 (2006.01) Camarillo, CA (US) HOIJ 49/00 (2006.01) GOIN3/84 (2006.01) (72) Inventors: Qi Huang, Camarillo, CA (US); Philip (52) U.S. C. A. Dimson, San Pedro, CA (US); CPC ...... G0IN30/7233 (2013.01); G0IN30/84 Karsten R. Liegmann, Altadena, CA (2013.01); H01J 49/0031 (2013.01); G0IN (US) 2030/8435 (2013.01); G0IN 2030/027 (73) Assignee: QUANTALYTICAL LABS INC., (2013.01) Camarillo, CA (US) (21) Appl. No.: 14/726,530 (57) ABSTRACT (22) Filed: May 30, 2015 The present specification discloses methods for determining Related U.S. Application Data the presence one or more analytes in a test sample, materials (60) Provisional application No. 62/005,676, filed on May useful to perform the disclosed methods, and kits comprising 30, 2014. reagents useful to practice the disclosed methods.

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EXTRACTION, DERIVATIZATION, AND estradiol, estriol), cannabinoids Such as A-9-tetrahydrocan QUANTIFICATION OF ANALYTES nabinol (THC), , amino acids, BPA, and many other primary and secondary containing, or phenol contain 0001. This application claims priority under 35 U.S.C. ing molecules in a biological sample. The desired analyte can S119(e) to U.S. Provisional Application 62/005,676 filed be selectively and sensitively detected and measured by mass May 30, 2014, the entire contents of which are hereby spectrometry, including tandem mass spectrometry technolo expressly incorporated by reference. gies. The entire quantification process includes a sample 0002. There is a need to obtain fast, streamlined, and auto preparation process that employs a Solid phase extraction to mated methods for detection of particular analytes. In the capture the analyte, followed by a chemical derivatization of medical context, such methods would facilitate the detection the analyte, then quantification via LC-MS/MS technologies, of analytes. Such as drugs, hormones, signaling agents, and as described herein. amino acids. In the agricultural and public health context, the 0007. It was discovered during this work that the combi detection of residual levels of antibiotics, pesticides or other nation of Solid phase extraction with a chemical derivatiza contaminants is integral to the safety of the food we eat and tion with Fmoc chloride and LC-MS/MS provides analyte water we drink. Furthermore, recent changes in the standards specific, sensitive, accurate, stable and robust measurements for food labeling, such as “hormone free” or “organic” have of levels of at 10 pg/ml or lower in blood created a need for streamlined testing in the agricultural plasma/serum sample. Quantification methods for metaneph realm. rines and thyroid hormones were also developed with similar 0003 Catecholamines are essential hormones or neu sensitivity and accuracy. rotransmitters that are important in maintaining a body’s 0008 Sensitive and accurate measurements of these ana healthy conditions. Neurological disorders such as Parkin lytes at levels relevant to the clinical setting is useful, particu son's disease, or Alzheimer's disease often may alter the larly at low pico-gram per milliliter range, at Small sample levels of these substances in the blood. Elevated levels of size (100 uL or less of blood plasma/serum sample), with a (derivatives of catecholamines) may be simple process which can be easily configured to be auto indicative of onset of certain cancers such as pheochromocy mated, with an analyte specific results, without any analyte toma. Similarly, metanephrines ( and normeta crossovers that are often seen under other analytical tech nephrine), thyroid hormones (thyroxine T4,3,5,5'-triiodothy niques such as radioimmunoassay (RIA). ronine T3, and 3,3',5-triiodothyronine rT3), estrogen hormones (estrone, esterol, estradiol, and estriol), delta-9- BRIEF DESCRIPTION OF THE DRAWINGS tetrahydrocannabinol (THC) its derivatives and metabolites, etc., are among many other biologically interesting molecular 0009 FIGS. 1A and 1B present example data showing the markers that are important for clinicians to determine the chromatograms for the internal standard equivalent 100 pg status of a patient. (FIG. 1A) and for the spiked plasma (FIG. 0004 Traditionally, clinical quantification of catechola 1B). mines were carried out via radio immunoassays (RIA). In (0010 FIGS. 2A and 2B represent data obtained at 10 and recent years, due to the technology advancement in Mass 100 pg/ml of each catecholamine. Spectroscopy, there is a gradual shift in testing platform from 0011 FIG. 3 provides the chromatograms for the meta RIA to liquid chromatography mass spectrometry (LCMS). nephrines: top being metanephrine (659.210/268.400), Often times, the LCMS based assays provide a fast, sensitive, middle being (645. 100/166.100) and lower and analyte specific readout, which an RIA assay may lack. being metanephrine (659.210/268.400). However, catecholamines are difficult to detect as they are (0012 FIGS. 4A, 4B, 4C, and 4D provide the spectra for prone to be oxidized and degraded. Further, the current art in the various shifts in metanephrine and normetanephrine. catecholamines/metanephrine quantification often involves (0013 FIGS.5A,5B,5C, and 5D provide the various peaks complex and cumbersome extraction procedures with at each shift for the Thyroid hormones: plasma with 5 ng/ml unstable extraction recovery, thus giving rise to results which equivalent (FIG. 5A), plasma blank (FIG. 5B), plasma 1 are often unreliable, and with high lower limits of quantifica ng/ml (FIG.5C), and 200 uL BSA with 1 ng/ml (FIG. 5D). tion (LLOQ); for example an LLOQ in >10 ng/mL range. (0014 FIGS. 6A, 6B, and 6C provide data for each of the Also, the existing extraction process is unable to provide a thyroid hormones T3 at 31.3 pg/ml (FIG. 6A), rT3 at 31.3 reliable sample for derivatization. pg/mL (FIG. 6B), and T4 at 31.3 pg/ml (FIG. 6C). 0005 Drugs include both illegal and legal drugs and (0015 FIGS. 7A, 7B, and 7C provide the linearity data for metabolites or derivatives thereof. The detection of these the quantification of 11-nor-9-carboxy-delta-9-tetrahydro compounds for forensic or prescription compliance are very cannabinol in Urine; 1,000 pg/ml (FIG. 7A), 100 pg/ml (FIG. important. Again, these drugs may not be stable in bodily 7B), and 10 pg/ml (FIG.7C). fluids and therefore, detection may be difficult. In any event, (0016 FIGS. 8A and 8B provide recovery data from the there is a great need for a fully automated and simplified quantification of 11-nor-9-carboxy-delta-9-tetrahydrocan detection/quantification procedure which minimizes the nabinol in Urine: Recovery-1,000 pg/rxn of Standard (FIG. room for human involvement or error. 8A)and Recovery of 1,000 pg/rxn Extracted Spike (FIG.8B). SUMMARY DETAILED DESCRIPTION 0006. This invention document provides materials and 0017. The present methods are directed to methods of methods that can be used measure the levels of catechola extraction, derivatization, and detection and/or quantification mines (, epinephrine, ), metaneph of analytes from a sample. The present methods employ a rines (metanephrine and normetanephrine), thyroid hor sample clean-up process via Solid phase extraction (SPE), mones (T4, T3, and rT3), estrogen hormones (estrone, chemical derivatization process in conjunction with detection US 2015/03461 70 A1 Dec. 3, 2015 or quantification of analytes of interest to obtain analyte spe moiety. An analyte as disclosed herein may be a drug (illegal cific, robust, fast, sensitive, and accurate results. The deriva and FDA approved) and a derivative or a metabolite thereof, tization of the analyte of interest shifts the analyte out of the a pesticide and a derivative or metabolite thereof, an environ region where it might potentially overlap with other biologi mental contaminant and a derivative or metabolite thereof, or cal agents found in the sample. Thus, during detection, other a biologic compound Such as, e.g., a hormone, a cytokine, a biological molecules or contaminants which would ordinarily signaling agent, an amino acid, cholesterol or one of its interfere with the accurate quantification of the analyte of derivatives, a fatty acid or a glycolipid, and a derivative or interest are detected at a significantly different range of the metabolite thereof. spectrum (or eluted completely), allowing the accurate quan 0022. In one embodiment, an analyte is a monoamine tification of the analyte of interest. Such analyte detection is , or one of its derivatives or metabolites. useful, e.g., in a clinical, a veterinarian, a forensic, and/or an Examples of monoamines include catecholamines Dopam environmental setting. ine (DA), Epinephrine (EP), Norepinephrine (NEP), or its 0018. The present methods may include derivatization of derivatives or metabolites: metanephrines Metanephrine the analytes of interest, and determination of the amount of (MNE), Normetanephrine (NMN), other trace one or more of the analytes by at least one of chromatography 3-Methoxytyramine (3-MT), p-Octopamine, Synephrine, and/or mass spectrometry. In one embodiment, the present Tyramine, 3,4-dihydroxybenzylamine, 3,4-Dihydroxyman methods may also include a solid phase extraction (SPE) of delic acid, Dihydroxyphenylethylene glycol, DOPAL, the analyte from a biological sample. In one aspect, it is DOPAC, , . 3-Methoxy-4- recommended that cation exchange based SPE may be used hydroxyphenylglycol, MOPET, or Normetanephrine, Vanil for capture amine containing analytes. For non-amine con lylmandelic acid, Catechol, Dopa, histamine, serotonin taining analytes, a reverse phase silica sorbent based SPE may (5-HT), 3-iodothyronamine, beta-phenethylamine, N-meth be employed with a protein crash with or without a phospho ylphenethylamine, tryptamine, N-methyltryptamine. lipid removal process, where such sorbent may be C4-C18 0023. In another embodiment, an analyte is an estrogen alkyl bounded silica, or phenyl bounded silica, or biphenyl including Estrone (E1), Estradiol (E2), Estriol (E3), and bonded silica. In other embodiments the final elution buffer Estetrol (E4). with high pH (8spHs 14) may be used to elute the amine 0024. In another embodiment, an analyte is a phytoestro containing analyte(s) from the cation exchange solid phase gen, including daidzein, formononetin, genistein, biochanin and optionally in situ derivatization of the amine containing A, coumestrol. 4'-methoxycoumestrol, repenSol, trifoliol, or analyte(s) of interest, cleanly preparing the derivative of the 17-beta-estradiol. analyte for detection and/or quantification. 0025. In another embodiment, an analyte is a cannabinoid 0019. In additional embodiments, the non-amine contain or one of its derivatives or metabolites. Examples of cannab ing analytes may be elute off from the SPE sorbent via a inoids or one of its derivatives or metabolites include a Can non-alcoholic, water miscible, organic solvent, such as aceto nabigerol-type (CBG) cannabinoid such as, e.g., Cannabig nitrile, DMF, acetone, 1,4-dioxane, THF, NMP, DMSO, and erol, Cannabigerol monomethyl ether, Cannabinerolic acid etc., followed by direct derivatization of the analyte under a A, Cannabigerovarin, Cannabigerolic acid A, Cannabigerolic pH buffered condition. In a preferred embodiment, the acid A monomethyl ether, and Cannabigerovarinic acid A.; a present methods for detection of an amine containing analyte Cannabichromene-type (CBC) cannabinoid, Such as, e.g., include cation exchange SPE, high pH elution of amine ana (t)-Cannabichromene, (+)-Cannabichromenic acid A, (+)- lyte from cation exchange sorbent, direct quantification via Cannabivarichromene, (+)-Cannabichromevarin, or (t)-Can LC-MS/MS or in situ derivatization, and then quantification nabichromevarinic acid A.; a Cannabidiol-type (CBD) can via LC-MS of the analyte of interest; permitting inline tran nabinoid such as, e.g., (-)-Cannabidiol, Cannabidiol sitioning from biological sample to initial analyte extraction momomethyl ether, Cannabidiol-C4, (-)-Cannabidivarin, to detection and/or quantification. Similarly, for the preferred Cannabidiorcol, Cannabidiolic acid, Cannabidivarinic acid; a embodiment for detection of a non-amine containing analyte, Cannabinodiol-type (CBND) cannabinoid Such as, e.g., Can present methods include reverse phase based SPE, organic nabinodiol or Cannabinodivarin; a Tetrahydrocannabinol elution followed by direct derivatization under high pH buffer type (THC) cannabinoid such as, e.g., A9-Tetrahydrocannab conditions, then completed by LC-MS/MS quantification. Or inol, A9-Tetrahydrocannabinol-C4, in another embodiment, an anion exchange based SPE may be A9-Tetrahydrocannabivarin, A9-Tetrahydrocannabiorcol, used for detection of an analyte with a carboxylic function A9-Tetrahydro-cannabinolic acid A. A9-Tetrahydro-cannab group, with a high pH elution buffer mixed with suitable inolic acid B. A9-Tetrahydro-cannabinolic acid-C4 A. organic solvents such as acetonitrile, DMF, acetone, 1,4- A9-Tetrahydro-cannabinolic acid-C4 B. A9-Tetrahydro-can dioxane, THF, NMP, DMSO, and etc., followed by direct nabivarinic acid A. A9-Tetrahydro-cannabiorcolic acid A. derivatization of the analyte under a pH buffered condition. A9-Tetrahydro-cannabiorcolic acid B, (-)-A8-trans-(6aR, 0020. Also presented are kits for a detection and/or quan 10aR)-A8-Tetrahydrocannabinol, (-)-A8-trans-(6aR.10aR)- tification assay. Such kits may include SPE columns (car Tetrahydrocannabinolic acid A, (-)-(6aS,10aR)-A9-Tetrahy tridges) and a derivatization reagent. Optionally, such kits drocannabinol; a Cannabinol-type (CBN) cannabinoid such may also include a SPE conditioning solvent, loading buffer, as, e.g., Cannabinol, Cannabinol-C4, Cannabivarin, Cannab washing buffer, and an eluent buffer, separately or together, inol-C2, Cannabiorcol, Cannabinolic acid A, Cannabinol and an HPLC column. methyl ether; a Cannabitriol-type (CBT) cannabinoid such 0021. The present methods may detect the presence of a as, e.g., (-)-(9R,1OR)-trans-Cannabitriol, (+)-(9S.10S)-Can wide array of analytes. An analyte is any compound or com nabitriol, (+)-(9R,10S/9S,10R)-Cannabitriol, (-)-(9R,10R)- position of interest to be found in the sample of interest. More trans-10-O-Ethyl-cannabitriol, (+)-(9R. 10R/9S.10S)-Can specifically, an analyte of interest is a compound having pri nabitriol-C3, 8.9-Dihydroxy-A6a(10a)- mary and or secondary amine groups, and or, a phenolic tetrahydrocannabinol, Cannabidiolic acid A cannabitriol US 2015/03461 70 A1 Dec. 3, 2015 ester, (-)-(6aR.9S. 10S,10aR)-9,10-Dihydroxy-hexahydro patient; that is, a living person, male or female, presenting cannabinol, (-)-6a, 7.10a-Trihydroxy-A9-tetrahydrocannab oneselfin a clinical setting for diagnosis, prognosis, or treat inol, 10-Oxo-A6a(10a)-tetrahydrocannabinol; a Cannabiel ment of a disease or condition. The sample is preferably soin-type (CBE) cannabinoid such as, e.g., (5aS,6S-9R.9aR)- obtained from a patient, for example, a plasma specimen. The Cannabielsoin, (5aS,6S-9R.9aR)-C3-Cannabielsoin, (5aS, plasma specimen may be taken with or without the use of 6S,9R,9aR)-Cannabielsoic acid A, (5aS,6S,9R,9aR)- anticoagulants. Cannabielsoic acid B, (5aS,6S-9R.9aR)-C3-Cannabielsoic 0033 Such biological samples may be obtained, for acid B, Cannabiglendol-C3, Dehydrocannabifuran, or Can example in a veterinarian setting, from an animal; that is, a pet nabifuran; an Isocannabinoid such as, e.g., (-)-A7-trans-(1R, animal, or a farm animal or livestock, a fish, or any other 3R,6R)-Isotetrahydrocannabinol, (+)-A7-1,2-cis-(1R,3R,6S/ creatures that live infreshwater, oceanor sea, male or female, 1S.3S,6R)-Isotetrahydrocannabivarin, or (-)-A7-trans-(1R, presenting oneself in a veterinarian setting for diagnosis, 3R,6R)-Isotetrahydrocannabivarin; a Cannabicyclol-type prognosis, prevention, or treatment of a disease or condition. (CBL) cannabinoid such as, e.g., (+)-(1aS.3aR.8bR.8cR)- The sample is preferably obtained from an animal, for Cannabicyclol. (+)-(1aS.3aR.8bR,8cR)-Cannabicyclolic example, a plasma specimen. The plasma specimen may be acid A, or (+)-(1aS.3aR.8bR.8cR)-Cannabicyclovarin; a taken with or without the use of anticoagulants. Cannabicitran-type (CBT) cannabinoid such as, e.g., Can 0034. A test sample may be obtained from a plant or any nabicitran; and a Cannabichromanone-type (CBCN) cannab Vegetation source, in agricultural or environmental setting, inoid such as, e.g., Cannabichromanone, Cannabichro from a leaf, or a flower, or a stem, or a fruit, or a seed, or manone-C3, or Cannabicoumaronone. sprout, or a bark, or a root, etc. 0026. In another embodiment, an analyte is a thyroid hor 0035 A test sample may be obtained from a dead human mone or one of its derivatives or metabolites. Examples of body, or a dead animal, or a dead plant, or remains of a once thyroid hormones or one of its derivatives or metabolites living body, as in a forensic setting, or in an agricultural include 3.3',5-triiodothyronine (T3), 3.5.5'-triiodothyronine setting, or in an environmental setting, or in an archeological (rT3), and thyroxine (T4). setting. A testing sample may be a blood sample, or a dry 0027. In another embodiment, an analyte is an opiate, blood sample, or any other body fluid sample, or any other dry opioid or one of its derivatives or metabolites. Examples, of body fluid sample, or a body tissue sample taken from any opiates or opioids or one of its derivatives or metabolites where of the body or remains of a dead human, animal, or include the naturally-occurring benzylisoquinoline alkaloids plant. (morphine, and oripavine), the semi-synthetic derivatives 0036. A test sample may be an environmental sample. (hydromorphone, and oxymorphone), and the synthetic opio Environmental samples are samples taken from dirt, plant ids (e.g., buprenorphine, etorphine, pentazocine). matter, or fluid sources (such as ground water, oceans, or 0028. In another embodiment, an analyte is arylcyclo rivers etc.). Dirt (aka “soil samples') may be taken from hexylamine or one of its derivatives or metabolites. Examples agricultural sites or sites of environmental interest and may of an arylcyclohexylamine include Tiletamine, 3-Methox have the analyte extracted, including the removal of particu etamine (MXE), Methoxyketamine, N-ethylnorletamine late matter. (Ethketamine) 0037 Samples may be obtained by any known means. The 0029. In another embodiment, an analyte is an Amphet sample may be preserved or pre-treated to ensure stability of amine. Examples of Amphetamines are Amphetamine (it the analyte of interest. Such preservation may be accom self), methamphetamine, ephedrine, cathinone, 3.4-methyl plished by chemical (Such as hydrolysis or pH adjustment) or enedioxy-N-methylamphetamine (MDMA, "Ecstasy”), and physical processes (such as refrigeration or freezing). When a 2.5-Dimethoxy-4-methylamphetamine (DOM, or "STP"). sample is a solid or a tissue, it can be grounded, or extracted, 0030. In another embodiment, an analyte is an amino acid, or purified, or filtered, or centrifuged, to isolate the analyte of an artificial amino acid, or a small peptide. Examples of the interest from the interfering components. Or a sample is a amino acid include but are not limited to: glycine, alanine, liquid, preferably, it is dissolved, or Suspended, in a solution phenylalanine, , GABA, , cysteine, serine, (or “loading buffer') having a pH range from weakly basic to Valine, leucine, isoleucine, lysine, methionine, histidine, argi neutral to weakly acidic; for example having a pH ranging nine, aspartic acid, asparagine, glutamic acid, glutamine, pro from 10-3, or more preferably 9-4, or more preferably 8-5, or line, and threonine. even more preferably 7-6, depending on the analyte of interest 0031. In another embodiment, an analyte is bisphenol A and the sorbent chemistry. (BPA). 0038 Any sample volume may be obtained as long as it is 0032. Aspects of the present specification disclose, in part, of sufficient volume to be useful in the methods disclosed a test sample. A test sample refers to any sample that may herein. In aspects of this embodiment, a sample Volume may contain an analyte of interest. A test sample may be a biologi be e.g., about 10 uL, about 25 uL, about 50 uL, about 75 uL. cal sample, that is, a sample obtained from any biological about 100 uL, about 125 uL, about 150 uL, about 175 uL. Source. Such as an animal, a plant, a fungus, a microorganism, about 200 uL, about 225 uL, about 250 uL, about 275 uL. a cell culture, an organ culture, etc. In aspects of this embodi about 300 uL, about 325 uL, about 350 uL, about 375 uL. ment, a biological sample includes a blood sample including about 400 uL, about 425 uL, about 450 uL, about 475 uL, or a whole blood sample, a dry blood sample, a plasma sample, about 500 uL. In other aspects of this embodiment, a sample or a serum sample, a saliva sample, a lachrymal sample, a Volume may be e.g., at least 10 uL. at least 25 LIL, at least 50 semen sample, a urine sample, cerebrospinal fluid sample, a uL, at least 75uL, at least 100LL, at least 125 uL, at least 150 bile sample, an embryonic fluid sample, a tissue sample, or uL, at least 175uL, at least 200LL, at least 225 uL, at least 250 any other sample that can be obtained, extracted or isolated uL, at least 275 uL, at least 300 uL, at least 325 uL, at least 350 from a biological source. Such biological samples may be uL, at least 375 uL, at least 400 uL, at least 425uL, at least 450 obtained, for example in a medical or clinical setting, from a uL, at least 475 uL, or at least 500 uL. In yet other aspects of US 2015/03461 70 A1 Dec. 3, 2015

this embodiment, a sample Volume may be e.g., at most 10LL. Solid phase, allowing undesired components of the mobile at most 25 uL, at most 50 uL, at most 75 uL, at most 100 uL. phase to pass through or around the Solid phase. In these at most 125 uL, at most 150 uL, at most 175 uL, at most 200 instances, a second mobile phase is then used to elute the LL, at most 225 uL, at most 250 uL, at most 275 uL, at most retained analyte off of the solid phase for further processing 300 uL, at most 325 uL, at most 350 uL, at most 375 uL, at or analysis. most 400 uL, at most 425ul, at most 450 uL, at most 475 uL. 0043 SPE using an ion exchange extraction procedure is or at most 500 uL. In still other aspects of this embodiment, a applied to extract the analytes of interest from the sample. sample Volume may be between e.g., about 10LL and about at Such analyte can be ionized under certain ranges of pH of a buffer. SPE may be performed with a range of characteristics most 100 uL, about 10 LIL and about at most 200 uL, about 10 Suitable depending on the analyte. Analytes such as monoam uL and about at most 300 uL, about 10 uL and about at most ine , or catecholamines, or metanephrines, 400 uL, about 10 uL and about at most 500 uL, about 10 ul or amino acids, or thyroid hormones, or carboxylic acids, and about at most 600 uL, about 10 LIL and about at most 700 maybe extracted, or retained, or purified, via ion exchange uL, about 10 uL and about at most 800 u, about 10 uL and extraction based SPE. More specifically strong to weak cation about at most 900 uL, about 10LL and about at most 1000 uL. exchange may be used. SPE using a cation exchange is one about 50 uL and about at most 100LL, about 50 uL and about example applied in the present methods of extracting analyte at most 200 uL, about 50 uL and about at most 300 uL, about of interest from a blood plasma sample. Weak cation 50 uL and about at most 400 uL, about 50 uL and about at exchange cartridges with a divinylbenzene- (DVB-) based most 500 uL, about 50 uL and about at most 600 uL, about 50 polymer sorbent are particularly exemplified for the SPE of uL and about at most 700 uL, about 50 uL and about at most catecholamines and metanephrines. SPE using a strong cat 800 uL, about 50 uL and about at most 900 uL, about 50 LL ion exchange extraction based SPE may also be used to purify and about at most 1000 uL, about 100 uL and about at most catecholamines and metanephrines from the blood plasma 200 u, about 100 uL and about at most 300 uL, about 100 uL sample with an SPE cartridge filled with a DVB-based poly and about at most 400 uL, about 100LL and about at most 500 mer sorbent via stronger elution solvent. Moreover, silica uL, about 100LL and about at most 600 uL, about 100 u, and based carboxylic acid sorbents may also be useful to extract about at most 700 uL, about 100LL and about at most 800 uL. catecholamines and metanephrines from the plasma samples. about 100 uL and about at most 900 uL, or about 100 uL and 0044 Astrong cation exchange (sulfonic acid chemistry) about at most 1000 uL. sorbent either based on silica or one or more polymers may 0039. A test sample disclosed herein may be purified. As also be useful to extract catecholamines and metanephrines in used herein, the terms “purified”, “purification” or “purify the SPE process. SPE with a strong cation exchange sorbent ing does not refer to removing all materials from the sample are particularly exemplified for the SPE of thyroid hormones other than the analyte(s) of interest. Instead, purification (T3/rT3/T4). An amino acid analyte may be similarly refers to a procedure that enriches the amount of one or more extracted with a strong cation exchange sorbent, either poly analytes of interest relative to other components in the sample merbased, or silica based. that may interfere with detection of the analyte of interest. 0045 An anion exchange polymer may also be used to Purification of the sample by various means may allow rela extract a carboxylic acid analyte, or an amino acid analyte, or tive reduction of one or more interfering Substances, e.g., one a Sulfonic acid analyte, or a phosphonic acid analyte. or more Substances that may or may not interfere with the 0046 Particular columns of interest for use in the present detection of selected parent or daughter ions of the selected methods to extract catecholamines and metanephrines analyte by mass spectrometry. Relative reduction as this term include the CEREX(R) PWCX, 1 mL Columns, 10 mg., 96/Pk is used does not require that any Substance, present with the (catalog number 6750-0101R. For thyroid hormones the analyte of interest in the material to be purified, is entirely CEREX(R) PSCX, 1 mL Columns, 10 mg. 96/Pk (catalog removed by purification. When detecting some analytes (par number 687-0101R) are suitable. ticularly drugs) in a urine sample, hydrolysis may be neces 0047. In yet another embodiment, a reverse phase silica sary to remove the glucoronide bonding which prevents the based SPE column or cartridge may be used to extract, or solubility and extraction of the analyte. This purification tech purify, or retain the analyte of choice when the analyte does nique is usually performed by enzyme or acid hydrolysis of not have an amino group, Such as estrogen hormones, or the urine. Alternatively, removing particulate matter (e.g., by cannabinoids, or flavonoids. The sorbent of choice during this centrifugation or filtration), protein precipitation (optionally SPE/purification may include alkyl bounded silica (C4, C8, by a “protein crash' method) with or without phospholipid C12, and C18), cyano bounded silica, phenyl bounded silica, removal, may be useful purification techniques. or biphenyl bounded silica. 0040 Purification may also be performed to create or 0048 Sizes of the columns may range, but in particular, a make available reactive amino or phenolic groups, suitable column volume may preferably range from 50 u to 3000 uL. for the derivatization reaction. These methods include with a sorbent loading between 100 ug to 50 mg. In another hydrolysis of esters or amines, or acid hydrolysis of Sugars. embodiment, the more preferred column size ranges from 100 0041. Such purification by pre-processing is not limited, uL to 2000 uL with a sorbent loading between 1 mg to 20 mg. but serves to prepare the sample for Solid phase extraction. In another embodiment, the more preferred column size 0042. As used herein, the term "solid phase extraction” or ranges from 200 uL to 1000 uL with a sorbent loading “SPE' refers to a process in which a chemical mixture is between 2 mg to 10 mg. Shape and size of the SPE columns separated into components as a result of the affinity of com (cartridges) may be varied to fit a specific platform. ponents dissolved or Suspended in a solution (i.e., mobile 0049. The particle size of the sorbent may further assist in phase) for a solid through or around which the solution is the separation of the analyte of interest. In aspects of this passed (i.e., Solid phase). In some instances, as the mobile embodiment, a particle size of a sorbent may have a mean phase passes through or around the Solid phase, undesired diameter of, e.g., about 0.5um, about 1 um, about 5um, about components of the mobile phase may be retained by the solid 10um, about 15um, about 20 Lum, about 25um, about 30 um, phase resulting in a purification of the analyte in the mobile about 35um, about 40 um, about 45um, about 50 um, about phase. In other instances, the analyte may be retained by the 55um, about 60 Lim, about 65um, about 70 Lum, or about 75 US 2015/03461 70 A1 Dec. 3, 2015

um. In other aspects of this embodiment, a particle size of a aqueous buffer. An organic solvent may be acetonitrile, or sorbent may have a mean diameter of, e.g., at least 0.5um, at methanol, or ethanol, or isopropanol, or butanol, or diethyl least 1 um, at least 5um, at least 10 um, at least 15um, at least ether, or acetone, or 1,4-dioxane, or THF, or DMF, ethyl 20 um, at least 25um, at least 30 Jum, at least 35um, at least acetate, or methyl acetate, or ethyl formate, methyl formate, 40 Lim, at least 45um, at least 50 um, at least 55um, at least or a mixture of any of the above solvents. 60 um, at least 65um, at least 70 um, or at least 75um. In yet 0052. Upon loading and washing, the loaded cartridge other aspects of this embodiment, a particle size of a sorbent may be treated with an elution fluid directly, or dried first via may have a mean diameter of, e.g., at most 0.5um, at most 1 a stream of dry nitrogen, or another dry inner gas, passing um, at most 5um, at most 10 um, at most 15um, at most 20 through the cartridge. um, at most 25um, at most 30 Jum, at most 35um, at most 40 0053 Aspects of the present specification disclose, in part, um, at most 45um, at most 50 um, at most 55um, at most 60 a method of in situ derivatization of the analyte of interest um, at most 65um, at most 70 um, or at most 75 um. In still using a derivatizing agent. The analyte of interest reacts with other aspects of this embodiment, a particle size of a sorbent a derivatizing reagent to provide a derivative of the analyte. may have a mean diameter in the range of, e.g., about 0.5um This derivative displays an improved HPLC behavior, and to about 10um, about 0.5um to about 20 um, about 0.5um to significantly improved tandem MS/MS sensitivity. For about 30 um, about 0.5um to about 40 um, about 0.5um to instance the present method unexpectedly provides nearly, or about 50 lum, about 0.5um to about 60 Lum, about 0.5um to over 1000-fold improvement in detection limitation and/or about 70 um, about 0.5 um to about 80 um, about 1 um to quantification sensitivity when quantifying catecholamines, about 10 Jum, about 1 um to about 20 um, about 1 um to about metanephrines, thyroid hormone T3, rT3, and T4, from blood 30 um, about 1 um to about 40 um, about 1 um to about 50 um, serum samples, by using LC-tandem MS/MS technologies. about 1 um to about 60 um, about 1 um to about 70 um, about 1 um to about 80 um, about 5um to about 10 um, about 5um 0054. A derivatizing reagent disclosed herein is a com to about 20 um, about 5 um to about 30 Lum, about 5 um to pound that can react with a primary or secondary amino, about 40 um, about 5um to about 50 m, about 5um to about and/or a phenolic hydroxy group, and/or a primary alcohol 60 um, about 5um to about 70 um, about 5um to about 80 um, (hydroxyl) group, and/or an aryl or alkylthiol group, present about 10 um to about 20 um, about 10 um to about 30 um, in an analyte disclosed herein. Derivatizing agents of interest include, without limitation, an acylating agent, as in Formula about 10 um to about 40 um, about 10 um to about 50 um, I. Acylating reagents may include acyl chlorides or other acyl about 10 um to about 60 um, about 10 Lum to about 70 um, or halides. Derivatizing agents may also fluoresce or participate about 10 um to about 80 Lum. in a colorimetric reaction to assist with the detection of the 0050. A sample may be loaded on the SPE column with a loading solvent, or a loading buffer. The loading solvent may bound analyte. be deionized water, or a pH buffered aqueous solution, or an organic solvent, or a mixture of organic solvents, or a mixture (I) of an organic solvent and deionized water, or a mixture of O organic solvents with deionized water, or a pH buffered aque ous Solution mixed with an organic Solvent or a mixture of us organic Solvents. The pH buffered aqueous Solution may be a A X phosphate buffered saline (PBS), or a phosphate buffer, or a carbonate buffer, or a succinate buffer, or a tartrate buffer, or 0055. In one embodiment, the derivatization is performed a citric buffer, or a formic buffer, or an acetic buffer, or using FMoc chloride and variants thereof. The word “vari another commonly used buffer Solutionina typical biochemi ants' is applied here to distinguish variations of FMoc chlo cal lab, or a mixture of any of the two, or more of the follow ride, examples thereof are partially represented in the For ing, a phosphate buffer, or a carbonate buffer, or an acetic mula II. It is to be understood that a variety of variants of buffer, or a formic buffer, or a citric buffer, or a succinate FMoc chloride are suitable for derivatization of the analyte buffer, or a tartrate buffer, or, with a pH range from weakly according to the present methods. Thus, the specific embodi basic to neutral to weakly acidic; for example having a pH ments discussed below are not an exclusive listing. ranging from 10-3, or more preferably 9-4, or more prefer 0056. In one embodiment, the present methods employ a ably 8-5, or even more preferably 7-6, at a concentration FMoc Chloride of Formula II: range from 0.1 mM to 100 mM, or more preferably 0.5 mM to 50 mM, or more preferably 1 mM to 25 mM, or more prefer

ably 5 mM to 10 mM. An organic solvent may be selected (II) from acetonitrile, or acetone, or 1,4-dioxane, or DMF, or tetrahydrofuran (THF), or diethylether, or ethyl acetate, or methyl acetate, or ethyl formate, methyl formate, or a mixture of thereof. 0051. Upon loading the sample to the SPE column, the fluid is allowed to pass through the Sorbent, via gravity, or a vacuum pulling through a vacuum manifold, or a nitrogen or inert gas pressure push through a positive pressure manifold, with or without an air drying process. The sample loaded cartridge may be further washed with a solvent. The selection of the washing solvent may be deionized water, or a pH buffered aqueous solution, or organic solvent, or a mixture of organic solvents, or a mixture of organic solvents with an US 2015/03461 70 A1 Dec. 3, 2015 wherein each R', R. R. and Rare each independently H, a Rare each independently H, a halogen atom such as a fluo halogen atom Such as a fluorine, or a chlorine, or a bromine, rine, or a chlorine, or a bromine, or an iodine, a cyano group, or an iodine, a cyano group, an acetylene group, a propylene an acetylene group, a propylene group, a vinyl group, low group, a vinyl group, low alkyl (linear, branched, or cyclic alkyl (linear or branched or cyclic Cls alkyl), a Substituted C1-8 alkyl), a substituted vinyl group, a linear or branched or vinyl group, a linear or branched or cyclic Cls alkyl group. cyclic C1-8 alkoxy group. Examples of a linear or branched Examples of a linear or branched Cls alkyl include, e.g., Cs alkyl include, e.g., methyl, ethyl, propyl, butyl, cyclic methyl, ethyl, propyl, butyl, cyclic propyl, cyclic pentyl, hex ane, heptyl, and octyl etc. The fused ring system may be an all propyl, cyclic pentyl, hexane, heptyl, and octyl etc. carbocyclic or a heterocyclic aromatic system, Such as a naph 0057. In aspects of this embodiment, and FMOC chloride thalene, or a benzofuran, or an indole, with a 5-7 membered compound is: fused ring, or a nonaromatic all carbocyclic, or heterocyclic fused ring system such as 3,4-dihydro-1H-indene, or a 3.4. Scheme 1 dihydro-2H-chromene, with a 4-8 membered fused ring. The IV fused ring may be substituted when appropriate with one or more of a halogenatom such as a fluorine, or a chlorine, or a O bromine, or an iodine, a cyano group, an acetylene group, a propylene group, a vinyl group, low alkyl (linear or branched or cyclic Cls alkyl), a Substituted vinyl group, a linear or r) 1. branched or cyclic Cls alkoxy group. Examples of a linear or branched Cls alkyl include, e.g., methyl, ethyl, propyl, butyl, cyclic propyl, cyclic pentyl, hexane, heptyl, and octyl etc.

Scheme 2 Chloroformic acid 9H-fluoren-9-ylmethyl ester O V 1s,

O CBZ Chloride r) 1s. R1 O R2 O lsC

R3 R5 R4 Substituted CBZ Chloride (2,7-di-tert-butyl-9H-fluoren-9-yl) RI O RI O methyl carbonochloridate VI R2 O ul C R2 O lsC R7 R7

R3 R4 O R6 R3 R4 O R6 ---, R5 R5 Substituted Fused Ring CBZ Chloride ( ) RI O RI O R2 O l C R2 O l C

(2,7-dibromo-9H-fluoren-9-yl) R3 R7 methyl carbonochloridate 0058 Multiple variants of FMoc chloride such as repre sented above Scheme 1 may be used as a parallel derivatiza Substituted Fused Ring CBZ Chloride tion reagent panel, to provide opportunities for simulta 0060. In another embodiment, as shown in Scheme 3, the neously quantifying multiple samples in one LCMS run. derivatization reagent may be a benzoyl chloride, or a variant 0059. In another embodiment, the derivatizing reagent thereof. A variant of benzoyl chloride is benzoyl chloride with may be benzyl carbonyl chloride (CBZ chloride), or a variant Substitutions on the phenyl ring, or a Substituted benzoyl thereof (Scheme 2). A variant is a CBZ chloride with substi chloride with a fused ring system, wherein each R',R,R,R' tutions on the phenyl ring, or a substituted CBZ chloride with , R. R. and Rare eachindependently a Hydrogen, a halogen a fused ring system, wherein each R', R. R. R. R. R. and atom Such as a fluorine, or a chlorine, or a bromine, or an US 2015/03461 70 A1 Dec. 3, 2015 iodine, a cyano group, an acetylene group, a propylene group. each independently a Hydrogen, a halogen atom such as a a vinyl group, low alkyl (linear or branched or cyclic C fluorine, or a chlorine, or a bromine, or an iodine, a cyano alkyl), a Substituted vinyl group, a linear or branched or cyclic group, an acetylene group, a propylene group, a vinyl group. Cs alkyl group. Examples of a linear or branched Cls alkyl include, e.g., methyl, ethyl, propyl, butyl, cyclic propyl. low alkyl (linear or branched or cyclic Cls alkyl), a Substi cyclic pentyl, hexane, heptyl, and octyl etc. The fused ring tuted vinyl group, a linear or branched or cyclic C1-8 alkyl system may be an all carbocyclic or a heterocyclic aromatic group. Examples of a linear or branched Cls alkyl include, system, Such as a naphthalene, or a benzofuran, or an indole, e.g., methyl, ethyl, propyl, butyl, cyclic propyl, cyclic pentyl, with a 5-7 membered fused ring, or a nonaromatic all car hexane, heptyl, and octyl etc. In a fused ring system, the bocyclic, or heterocyclic 4-8 membered fused ring system system may be an all carbocyclic or heterocyclic aromatic such as 3,4-dihydro-1H-indene, or a 3.4.dihydro-2H system, Such as a naphthalene, or a benzofuran, or an indole, chromene. The fused ring may be substituted when appropri with a fused ring sized from 5-7 membered, or a nonaromatic ate with one or more of a halogenatom Such as a fluorine, or all carbocyclic, or heterocyclic fused ring system such as a chlorine, or a bromine, or an iodine, a cyano group, an 3,4-dihydro-1H-indene, or a 3.4.dihydro-2H-chromene, with acetylene group, a propylene group, a vinyl group, low alkyl a fused ring sized from 4-8 membered. The fused ring may be (linear or branched or cyclic Cls alkyl), a Substituted vinyl Substituted when appropriate one or more of a halogen atom group, a linear or branched or cyclic Cls alkyl group. Such as a fluorine, or a chlorine, or a bromine, or an iodine, a Examples of a linear or branched Cls alkyl include, e.g., cyano group, an acetylene group, a propylene group, a vinyl methyl, ethyl, propyl, butyl, cyclic propyl, cyclic pentyl, hex group, low alkyl (linear or branched or cyclic Cls alkyl), a ane, heptyl, and octyl etc. Substituted vinyl group, a linear or branched or cyclic Cs alkyl group. Examples of a linear or branched Cls alkyl Scheme 3 include, e.g., methyl, ethyl, propyl, butyl, cyclic propyl. O cyclic pentyl, hexane, heptyl, and octyl etc.

C

Benzoyl Chloride R O

Benzene Sulfonyl Chloride RI O. O 2 VW R Sn C Substituted Benzoyl Chloride RI O R1 O R3 R5 R2 R2 R4 C C R7 R7 Substituted Benzene Sulfonyl Chloride RI

R3 R4 O R6 R3 R4 O R6 R5 R5 Substituted Fused Ring Benzoyl Chloride RI O R1 O R2 R2 C C Substituted Fused Ring Benzene Sulfonyl Chloride R3 R7 R1 O O R" O O R2 Y R2 Y C C Substituted Fused Ring Benzoyl Chloride R3 R7 0061 Another embodiment of the derivatization reagent as shown in Scheme 4 may be a benzenesulfonyl chloride, and a variant thereof. A variant of a benzenesulfonyl chloride may be a benzenesulfonyl chloride with substitutions on the ben Substituted Fused Ring Benzene Sulfonyl Chloride Zene ring, or a substituted benzenesulfonyl chloride with a fused ring system, wherein R. R. R. R. R. R. and R7 are US 2015/03461 70 A1 Dec. 3, 2015

0062. In another embodiment of the acylating reagent, it -continued may be an acyl bromide, oran acyl fluoride, or a 4-nitrophenol ester, or a pentafluorophenol ester, or an acylimidazole, or a hydroxySuccimide ester (OSu), or a hydroxySuccimide O sodium sulfonate ester (sulfo OSu), or a hydroxybenzotrizole ester (OBt), or a hydroxyl-aza-benzotriazole ester (OAt). The activated acyl reagents may be formed prior to the derivati Zation reaction, or may also be formed in situ during the 1, Cl, derivatization reaction process via appropriate precursors and activation reagents that a skilled person will know. Acylating ( ) reagents provided in Scheme 5 are examples of the scope of the reagent, and not the limits to the scope. R R R

O O y-sV/

R NaN a N N Where X is one of the following:Cl, Br, F, ().N s o1 s F

F F N NO F O -/ \ O O N - O s F s O O SONa O o1 N o1 N O-N IO O s O s O O s Such acylating reagents may be formed prior to the derivatization, may also be formed in situ during the derivatization process from appropriate precursors and coupling reagents,

0063) Notably, multiple acylating reagents may be used in parallel fashion to derivatize the same analytes in multiple samples then combined into one LCMS quantification pro cess, so called multiplex analysis, to significantly improve instrument operational efficiency and save solvents for LC. These acylating reagents may be from the same chemical class or from different chemical classes, as discussed above. 0064. In situ derivatization is performed either just before elution, during elution, or right after elution, or from just before the elution continued until after the elution, from the solid phase extraction column. The elution is performed with a high pH elution solution. As used herein, “high pH includes elution solutions having a basic pH. In an aspect of this embodiment, an elution Solution may have a pH of, e.g., about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13. In other aspect of this embodiment, an elution solution may have a pH of, e.g., at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5, at least 12, at least 12.5, or at least 13. In yet other aspect of this embodi ment, an elution solution may have a pH of e.g., at most 8, at US 2015/03461 70 A1 Dec. 3, 2015

most 8.5, at most 9, at most 9.5, at most 10, at most 10.5, at 0067. An elution solution disclosed herein may be buff most 11, at most 11.5, at most 12, at most 12.5, or at most 13. ered using any buffer having an alkaline buffering capacity. In In yet other aspect of this embodiment, an elution Solution aspects of this embodiment, an elution Solution disclosed may have a pH in the range of, e.g., about 8 to about 9, about herein may be buffered using one or a mixture of the organic 8 to about 9.5, about 8 to about 10, about 8 to about 10.5, or inorganic buffering agents, e.g., POPSO, TEA, phosphate. about 8 to about 11, about 8 to about 11.5, about 8 to about 12, In other aspects of this embodiment, an elution solution dis closed herein may be buffered using, e.g., a trialkyl-ammo about 8 to about 12.5, about 8 to about 13, about 8.5 to about nium buffer comprising, e.g., trialkylammonium bicarbonate, 9, about 8.5 to about 9.5, about 8.5 to about 10, about 8.5 to trialkylammonium borate, trialkylammonium carbonate, or a about 10.5, about 8.5 to about 11, about 8.5 to about 11.5, trialkylammonium phosphate; a cesium buffer comprising, about 8.5 to about 12, about 8.5 to about 12.5, about 8.5 to e.g., cesium bicarbonate, cesium borate, cesium carbonate, about 13, about 9 to about 9.5, about 9 to about 10, about 9 to cesium hydroxide, or dibasic cesium phosphate, or tribasic about 10.5, about 9 to about 11, about 9 to about 11.5, about cesium phosphate; a potassium buffer comprising, e.g., 9 to about 12, about 9 to about 12.5, about 9 to about 13, about potassium bicarbonate, potassium borate, potassium carbon 9.5 to about 10, about 9.5 to about 10.5, about 9.5 to about 11, ate, potassium hydroxide, or dibasic potassium phosphate, or about 9.5 to about 11.5, about 9.5 to about 12, about 9.5 to tripotassium phosphate; a sodium buffer comprising, e.g., about 12.5, about 9.5 to about 13, about 10 to about 10.5, Sodium bicarbonate, sodium borate, Sodium carbonate, diba about 10 to about 11, about 10 to about 11.5, about 10 to about sic sodium phosphate, tribasic sodium phosphate, Sodium 12, about 10 to about 12.5, about 10 to about 13, about 10.5 to hydroxide, or sodium tetraborate; a tetraalkylammonium about 11, about 10.5 to about 11.5, about 10.5 to about 12, buffer, comprising, e.g., tetraalkylammonium bicarbonate, about 10.5 to about 12.5, about 10.5 to about 13, about 11 to tetraalkylammonium borate, tetraalkylammonium carbonate, about 11.5, about 11 to about 12, about 11 to about 12.5, or oratetraalkylammonium phosphate, in water, with or without about 11 to about 13. the use of one of more of the following organic co-solvents 0065. In another embodiment, the elution is performed such as acetonitrile, or acetone, or tetrohydrofuran (THF), or with a lower pH elution solution. As used herein, “low pH 1,4-dioxane, or dimethylformamide (DMF), or N-methyl includes elution solutions having an acidic pH. In an aspect of pyrrolidone (NMP), or dimethyl sulfoxide (DMSO), or hex this embodiment, an elution solution may have a pH of, e.g., amethylphosphoramide (HMPA), or diethyl ether, or isopro about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, pyl alcohol (IPA), ort-butanol, or 2-butanol, etc., in a desired about 5, about 5.5, about 6, or about 6.5. In other aspect of this ratio, such as at/below for above, 5%, or 10%, or 15%, or 20%, embodiment, an elution solution may have a pH of, e.g., at or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, 60%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, of at least 5, at least 5.5, at least 6, or at least 6.5. In yet other organic in water. aspect of this embodiment, an elution Solution may have a pH 0068. The amount of buffer used in an elution solution of e.g., at most 2, at most 2.5, at most 3, at most 3.5, at most may be any concentration that can effectively maintain the 4, at most 4.5, at most 5, at most 5.5, at most 6, or at most 6.5. alkaline buffering capacity of the buffer. In aspects of this In yet other aspect of this embodiment, an elution Solution embodiment, an effective concentration of buffer may be, may have a pH in the range of, e.g., about 2 to about 3, about e.g., about 1.0 mM, about 5.0 mM, about 10 mM, about 20 2 to about 3.5, about 2 to about 4, about 2 to about 4.5, about mM, about 30 mM, about 40 mM, about 50 mM, about 60 2 to about 5, about 2 to about 5.5, about 2 to about 6, about 2 mM, about 70 mM, about 80 mM, about 90 mM, about 100 to about 6.5, about 2.5 to about 3, about 2.5 to about 3.5, about mM, about 200 mM, about 300 mM, about 400 mM, about 2.5 to about 4, about 2.5 to about 4.5, about 2.5 to about 5, 500 mM, about 600 mM, about 700 mM, about 800 mM, or about 2.5 to about 5.5, about 2.5 to about 6, about 2.5 to about about 900 mM, or about 1 M. In other aspects of this embodi 6.5, about 3 to about 3.5, about 3 to about 4, about 3 to about ment, an effective concentration of buffer may be, e.g., at least 4.5, about 3 to about 5, about 3 to about 5.5, about 3 to about 1.0 mM, at least 5.0 mM, at least 10 mM, at least 20 mM, at 6, about 3 to about 6.5, about 3.5 to about 4, about 3.5 to about least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, 4.5, about 3.5 to about 5, about 3.5 to about 5.5, about 3.5 to at least 70 mM, at least 80 mM, at least 90 mM, at least 100 about 6, about 3.5 to about 6.5, about 4 to about 4.5, about 4 mM, at least 200 mM, at least 300 mM, at least 400 mM, at to about 5, about 4 to about 5.5, about 4 to about 6, about 4 to least 500 mM, at least 600 mM, at least 700 mM, at least 800 about 6.5, about 4.5 to about 5, about 4.5 to about 5.5, about mM, or at least 900 mM, or at least 1 M. In yet other aspects 4.5 to about 6, about 4.5 to about 6.5, about 5 to about 5.5, of this embodiment, an effective concentration of buffer may about 5 to about 6, about 5 to about 6.5, about 5.5 to about 6, be, e.g., at most 1.0 mM, at most 5.0 mM, at most 10 mM, at about 5.5 to about 6.5, or about 6 to about 6.5. most 20 mM, at most 30 mM, at most 40 mM, at most 50 mM, 0066. In another embodiment, the elution is performed at most 60 mM, at most 70 mM, at most 80 mM, at most 90 with a neutral pH elution solution. In an aspect of this mM, at most 100 mM, at most 200 mM, at most 300 mM, at embodiment, an elution solution may have a pH of, e.g., about most 400 mM, at most 500 mM, at most 600 mM, at most 700 6.5, about 7, about 7.5 or about 8. In another aspect of this mM, at most 800 mM, or at most 900 mM, or at most 1 M. embodiment, an elution solution may have a pH of, e.g., at 0069. In still other aspects of this embodiment, an effec least 6.5, at least 7, at least 7.5 or at least 8. In yet another tive concentration of elution buffer may be in the range of aspect of this embodiment, an elution Solution may have a pH e.g., about 0.1 mM to about 10 mM, about 0.1 mM to about 25 of e.g., at most 6.5, at most 7, at most 7.5 or at most 8. In yet mM, about 0.1 mM to about 50 mM, about 0.1 mM to about other aspect of this embodiment, an elution solution may have 75 mM, about 0.1 mM to about 100 mM, about 0.1 mM to a pH in the range of, e.g., about 6.5 to about 7, about 6.5 to about 200 mM, about 0.1 mM to about 300 mM, about 0.1 about 7.5, about 6.5 to about 8, about 7 to about 7.5, about 7 mM to about 400 mM, about 0.1 mM to about 500 mM, about to about 8, or about 7.5 to about 8. 0.1 mM to about 1000 mM, about 1 mM to about 10 mM, US 2015/03461 70 A1 Dec. 3, 2015

about 1 mM to about 25 mM, about 1 mM to about 50 mM, 15° C. to 70° C., more preferably from 20° C. to 60° C., more about 1 mM to about 75 mM, about 1 mM to about 100 mM, preferably from 20° C. to 50° C. about 1 mM to about 200 mM, about 1 mM to about 300 mM, 0073. A derivatization reaction is performed under time about 1 mM to about 400 mM, about 1 mM to about 500 mM, conditions suitable for the attachment of the derivatizing about 1 mM to about 1000 mM, about 5 mM to about 25 mM, agent to the analyte. In aspects of this embodiment, the about 5 mM to about 50 mM, about 5 mM to about 75 mM, derivatization reaction is performed at duration range from 1 about 5 mM to about 100 mM, about 5 mM to about 200 mM, minute to about 24 hours, more preferably from 3 minutes to about 5 mM to about 300 mM, about 5 mM to about 400 mM, about 5 hours, more preferably from 5 minutes to 60 minutes, about 5 mM to about 500 mM, about 5 mM to about 1000 more preferably from 10 minutes to 30 minutes. mM, about 10 mM to about 25 mM, about 10 mM to about 50 0074. Of course, changing the temperature may change mM, about 10 mM to about 75 mM, about 10 mM to about the reaction time. For instance, if the reaction is heated, for 100 mM, about 10 mM to about 200 mM, about 10 mM to example, to 40°C., reaction time may be shortened. about 300 mM, about 10 mM to about 400 mM, about 10 mM 0075. The derivatization reaction may be quenched by to about 500 mM, about 10 mM to about 1000 mM, about 25 addition of a buffered solution, which contains one or more of mM to about 50 mM, about 25 mM to about 75 mM, about 25 buffer agents. In one embodiment, the buffer is an ammonium mM to about 100 mM, about 25 mM to about 200 mM, about buffer, such as ammonium formate, or ammonium acetate, or 25 mM to about 300 mM, about 25 mM to about 400 mM, ammonium carbonate, or triammonium phosphate, or ammo about 25 mM to about 500 mM, about 25 mM to about 1000 nium Sulfate, or ammonium borate, ammonium hydroxide, mM, about 50 mM to about 75 mM, about 50 mM to about ammonium chloride, ammonium Sulfate, ammonium bicar 100 mM, about 50 mM to about 200 mM, about 50 mM to bonate, ammonium bisulfate, bisammonium phosphate, etc. about 300 mM, about 50 mM to about 400 mM, about 50 mM In another embodiment, the quenching reagent may also be a to about 500 mM, about 50 mM to about 1000 mM, about 75 buffered D-amino acid solution, Such as glycine, alanine, or a mM to about 100 mM, about 75 mM to about 200 mM, about buffered D-alanine, or a buffered primary amine solution 75 mM to about 300 mM, about 75 mM to about 400 mM, such as in a concentration range from 1 mM to 500 mM, in about 75 mM to about 500 mM, about 75 mM to about 1000 water, or a mixed solvent of water and an organic solvent, mM, about 100 mM to about 150 mM, about 100 mM to about Such as an alcohol, e.g., methanol, or ethanol, or propanol, or 200 mM, about 100 mM to about 300 mM, about 100 mM to butanol, or glycol, or glycerol, or etc., or acetonitrile, or about 400 mM, about 100 mM to about 500 mM, about 100 acetone, or ether, or THF, or 1,4-dioxanes, or DMF, or NMP, mM to about 1000 mM, about 200 mM to about 300 mM, or DMSO, or HMPA. The quenching process neutralizes about 200 mM to about 400 mM, about 200 mM to about 500 most of any excess derivatizing reagent that may remain in the mM, about 200 mM to about 1000 mM, about 250 mM to eluent solution and also stabilizes the product by lowering the about 300 mM, about 250 mM to about 400 mM, about 250 pH of the reaction mixture. In aspects of this embodiment, the mM to about 500 mM, or about 250 mM to about 1000 mM. pH of the eluent containing the derivatized analyte may be In another embodiment, an effective concentration of the lowered to a range from about 4 to about 9.5. elution buffer may be in the range of about 5 mM to about 250 0076. After quenching, the reaction mixture containing mM. derivatized analyte may then be directly analyzed for the 0070 An elution solution disclosed herein may comprise a presence of the analyte of interest. Such analysis may be derivatizing agent disclosed herein. The amount of derivatiz either qualitative or quantitative in nature. In aspects of this ing agent added to an elution solution disclosed herein is an embodiment, eluent containing derivatized analyte may be amount in Sufficient access to enable a complete derivatiza directly analyzed for the presence of the analyte of interest tion of the analyte of interest for subsequent detection. The using, e.g., chromatography and/or mass spectroscopic detec derivatizing reagent may be mixed with the elution buffer tion. prior to the elution as in an in situ derivatization process, it 0077. As used herein, the term "chromatography” refers to may also be added to the eluent right after the elution to a process in which a chemical mixture carried by a liquid or fashion a post elution derivatization process. gas is separated into components as a result of differential distribution of the chemical entities as they flow around or 0071. In one embodiment, the simultaneous elution and over a stationary liquid or Solid phase. derivatization of the analyte may be accomplished by apply 0078. As used herein, the term “liquid chromatography' ing to the analyte bound to a solid sorbent Support matrix a or “LC’ means a process of selective retardation of one or high pH elution buffer which includes a derivatizing agent. more components of a fluid solution as the fluid uniformly Upon application of the elution solution, a derivatization percolates through a column of a finely divided Substance, or reaction occurs that converts the analyte to its derivative and through capillary passageways. The retardation results from then the derivative is eluted off the sorbent matrix at the same the distribution of the components of the mixture between one time as the elution of the analyte from the solid support or more stationary phases and the bulk fluid, (i.e., mobile matrix. phase), as this fluid moves relative to the stationary phase(s). 0072 The derivatization reaction may be conducted under Examples of “liquid chromatography’ include reverse phase any condition Suitable for the acylation of the analyte. In one liquid chromatography (RPLC), high performance liquid embodiment, a derivatization reaction is performed under chromatography (HPLC), ultra-high pressure liquid chroma temperature conditions suitable for the attachment of the tography (UHPLC), and turbulent flow liquid chromatogra derivatizing agent to the analyte. In aspects of this embodi phy (TFLC) (sometimes known as high turbulence liquid ment, a derivatization reaction may be performed at a tem chromatography) (HTLC) or high throughput liquid chroma perature range from about or above 0°C. to about or below tography, or nano-flow liquid chromatography, or nano LC 100° C., more preferably from about 5° C. to about 90° C., 0079. As used herein, the term “high performance liquid more preferably from 10° C. to 80°C., more preferably from chromatography” or “HPLC refers to liquid chromatogra US 2015/03461 70 A1 Dec. 3, 2015

phy in which the degree of separation is increased by forcing face that interacts with the various chemical moieties to facili the mobile phase under pressure through a stationary phase, tate separation of the chemical moieties. One suitable bonded typically a densely packed column. Surface is a hydrophobic bonded surface Such as an alkyl 0080. As used herein, the term “turbulent flow liquid chro bonded, a cyano bonded, or a pentafluorophenylpropyl (F5) matography” or “TFLC' (sometimes known as high turbu surface, or phenyl/bonded, or biphenyl bonded surface. Alkyl lence liquid chromatography (HTLC) or high throughput liq bonded surfaces may include C-4, C-8, C-12, or C-18 bonded uid chromatography) refers to a form of chromatography that alkyl groups. In preferred embodiments, the column is a C-18 utilizes turbulent flow of the material being assayed through column. The chromatographic column includes an inlet port the column packing as the basis for performing the separa for receiving a sample directly or indirectly from a solid tion. TFLC has been applied in the preparation of samples phase extraction or HTLC column and an outlet port for containing two unnamed drugs prior to analysis by mass discharging an effluent that includes the fractionated sample. spectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: I0085. In certain embodiments, an analyte may be enriched 23-35 (1999); see also, U.S. Pat. Nos. 5,968.367, 5,919,368, in a sample by applying a sample to a column under condi 5,795,469, and 5,772,874, which further explain TFLC. Per tions where the analyte of interest is reversibly retained by the sons of ordinary skill in the art understand “turbulent flow”. column packing material, while one or more other materials When fluid flows slowly and smoothly, the flow is called are not retained. In these embodiments, a first mobile phase “laminar flow”. For example, fluid moving through an HPLC condition can be employed where the analyte of interest is column at low flow rates is laminar. In laminar flow the retained by the column, and a second mobile phase condition motion of the particles of fluid is orderly with particles mov can Subsequently be employed to remove retained material ing generally in straight lines. At faster Velocities, the inertia from the column, once the non-retained materials are washed of the water overcomes fluid frictional forces and turbulent through. Alternatively, an analyte may be enriched in a flow results. Fluid not in contact with the irregular boundary sample by applying a sample to a column under mobile phase “outruns that which is slowed by friction or deflected by an conditions where the analyte of interest elutes at a differential uneven surface. When a fluid is flowing turbulently, it flows in rate in comparison to one or more other materials. Such eddies and whirls (or vortices), with more "drag' than when procedures may enrich the amount of one or more analytes of the flow is laminar. Many references are available for assist interest relative to one or more other components of the ing in determining when fluid flow is laminar or turbulent sample. In another embodiment, the reaction mixture of the (e.g., Turbulent Flow Analysis Measurement and Prediction, analyte may be first loaded onto a guard column with a weak P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc., solvent to retain the desired analyte product on the guard (2000); An Introduction to Turbulent Flow, Jean Mathieu & column, then an LC elution solvent is to carry the Substrate Julian Scott, Cambridge University Press (2001)). onto the analytical column for separation and analysis. 0081. As used herein, the term “gas chromatography” or I0086. In one embodiment, the sample may be applied to “GC refers to chromatography in which the sample mixture the LC column at the inlet port, eluted with a solvent or is vaporized and injected into a stream of carrier gas (as solvent mixture, and discharged at the outlet port. Different nitrogen or helium) moving through a column containing a Solvent modes may be selected for eluting the analyte(s) of stationary phase composed of a liquid or a particulate Solid interest. For example, liquid chromatography may be per and is separated into its component compounds according to formed using a gradient mode, an isocratic mode, or a poly the affinity of the compounds for the stationary phase. typic (i.e. mixed) mode. During chromatography, the separa 0082. As used herein, the term “large particle column” or tion of materials is effected by variables such as choice of “extraction column” refers to a chromatography column con eluent (also known as a “mobile phase'), elution mode, gra taining an average particle diameter greater than about 50 um. dient conditions, temperature, etc. As used in this context, the term “about” meansit 10%. I0087. In one preferred embodiment, HPLC is conducted 0083. As used herein, the term “analytical column” refers with a hydrophobic column chromatographic system. In cer to a chromatography column having sufficient chromato tain preferred embodiments, a C18 analytical column (e.g., a graphic plates to effect a separation of materials in a sample TARGAR) C18, 3 Lum 50x2.1, or equivalent) is used. In certain that elute from the column sufficient to allow a determination preferred embodiments, HPLC are performed using HPLC of the presence or amount of an analyte. Such columns are Grade 5.0 mMammonium formate with 0.1% formic acid at often distinguished from “extraction columns, which have a pH of 3.0 and 0.1% formic acid in acetonitrile as the mobile the general purpose of separating or extracting retained mate phases. rial from non-retained materials in order to obtain a purified I0088. By careful selection of valves and connector plumb sample for further analysis. As used in this context, the term ing, two or more chromatography columns may be connected “about means-10%. as needed Such that material is passed from one to the next 0084 Certain methods of liquid chromatography, includ without the need for any manual steps. In preferred embodi ing HPLC, rely on relatively slow, laminar flow technology. ments, the selection of valves and plumbing is controlled by Traditional HPLC analysis relies on column packing in which a computer pre-programmed to perform the necessary steps. laminar flow of the sample through the column is the basis for Most preferably, the chromatography system is also con separation of the analyte of interest from the sample. The nected in Such an on-line fashion to the detector system, e.g., skilled artisan will understand that separation in Such col an MS system. Thus, an operator may place a tray of samples umns is a diffusional process and may select HPLC instru in an autosampler, and the remaining operations are per ments and columns that are Suitable for use with the analytes formed under computer control, resulting in purification and of interest. The chromatographic column typically includes a analysis of all samples selected. medium (i.e., a packing material) to facilitate separation of I0089. In some embodiments, the solid phase extraction chemical moieties (i.e., fractionation). The medium may may be used in a high throughput platform for enrichment of include minute particles. The particles include a bonded Sur the derivatized analyte of interest prior to mass spectrometry. US 2015/03461 70 A1 Dec. 3, 2015

In Such embodiments, samples may be extracted using a high ment where, due to a combination of magnetic and electric throughput SPE cartridge, or a guard column which captures fields, the ions follow a path in space that is dependent upon the derivatized analyte, then eluted onto an analytical HPLC mass (“m’) and charge (“Z”). See, e.g., U.S. Pat. No. 6,204. column, Such as a C-18 column, prior to mass spectrometry 500, entitled “Mass Spectrometry From Surfaces:” U.S. Pat. (MS) analysis. Because the steps involved in these chroma No. 6,107,623, entitled “Methods and Apparatus for Tandem tography procedures may be linked in an automated fashion, Mass Spectrometry:” U.S. Pat. No. 6,268,144, entitled “DNA the requirement for operator involvement during the purifi Diagnostics Based On Mass Spectrometry: U.S. Pat. No. cation of the analyte can be minimized. This feature may 6,124,137, entitled "Surface-Enhanced Photolabile Attach result in Savings of time and costs, and eliminate or reduce the ment And Release For Desorption And Detection Of Ana opportunity for an operator error. lytes: Wright et al., Prostate Cancer and Prostatic Diseases 0090 Direct quantification is “inline” or “on-line” use of 1999, 2: 264-76; and Merchant and Weinberger, Electro the extracted and derivatized analyte for quantification. As phoresis 2000, 21: 1164-67. used herein, the term “on-line' or “inline', for example as 0.095 As used herein, the term “electrospray ionization' used in “on-line automated fashion' or “on-line extraction' or "ESI. refers to methods in which a solution is passed along refers to a procedure performed without the need for operator a short length of capillary tube, to the end of which is applied intervention. In contrast, the term "off-line' as used herein a high positive or negative electric potential. Solution reach refers to a procedure requiring manual intervention of an ing the end of the tube is vaporized (nebulized) into a jet or operator. Thus, if samples are Subjected to precipitation, and spray of very small droplets of solution in solvent vapor. This the Supernatants are then manually loaded into an autosam mist of droplets flows through an evaporation chamber, which pler, the precipitation and loading steps are off-line from the may be heated to prevent condensation and to facilitate sol Subsequent steps. In various embodiments of the methods, vent evaporation. As the droplets get Smaller the electrical one or more steps may be performed in an on-line automated Surface charge density increases until Such time that the natu fashion. ral repulsion between like charges causes ions as well as 0091. As used herein, the term “sample injection” refers to neutral molecules to be released. In one embodiment, the introducing an aliquot of a single sample into an analytical detection is performed after ESI. instrument, for example a mass spectrometer. This introduc 0096. As used herein, the term "atmospheric pressure tion may occur directly or indirectly. An indirect sample chemical ionization' or APCI refers to mass spectrometry injection may be accomplished, for example, by injecting an methods that are similar to ESI; however, APCI produces ions aliquot of a sample into a HPLC column that is connected to by ion-molecule reactions that occur within a plasma at atmo a mass spectrometer in an on-line fashion. spheric pressure. The plasma is maintained by an electric 0092. As used herein, the term “same sample injection' discharge between the spray capillary and a counter elec with respect to multiple analyte analysis by mass spectrom trode. Then ions are typically extracted into the mass analyzer etry means that the molecular ions for two or more different by use of a set of differentially pumped skimmer stages. A analytes are determined essentially simultaneously by mea counterflow of dry and preheated N2 gas may be used to Suring molecularions for the different analytes from the same improve removal of solvent. The gas-phase ionization in (i.e. identical) sample injection. APCI can be more effective than ESI for analyzing less-polar 0093. In various embodiments, the analytes of interest species. present in a test sample may be ionized by any method known 0097. The term "atmospheric pressure photoionization' to the skilled artisan. Mass spectrometry is performed using a or “APPI as used herein refers to the form of mass spectrom mass spectrometer, which includes an ion source for ionizing etry where the mechanism for the photoionization of mol the fractionated sample and creating charged molecules for ecule M is photon absorption and electron ejection to form the further analysis. For example ionization of the sample may be molecularion M--. Because the photon energy typically is just performed by electron ionization, chemical ionization, elec above the ionization potential, the molecular ion is less Sus trospray ionization (ESI), photon ionization, atmospheric ceptible to dissociation. In many cases it may be possible to pressure chemical ionization (APCI), photoionization, atmo analyze samples without the need for chromatography, thus spheric pressure photoionization (APPI), fast atom bombard saving significant time and expense. In the presence of water ment (FAB), liquid secondary ionization (LSI), matrix vapor or protic solvents, the molecular ion can extract H to assisted laser desorption ionization (MALDI), field ioniza form MH--. This tends to occur if M has a high proton affinity. tion, field desorption, thermospray/plasmaspray ionization, This does not affect quantitation accuracy because the Sum of surface enhanced laser desorption ionization (SELDI), induc M+ and MH-- is constant. Drug compounds in protic solvents tively coupled plasma (ICP) and particle beam ionization. are usually observed as MH--, whereas nonpolar compounds The skilled artisan will understand that the choice of ioniza Such as naphthalene or testosterone usually form M--. See, tion method may be determined based on the analyte to be e.g., Robb et al., Anal. Chem. 2000, 72(15): 3653-3659. measured, type of Sample, the type of detector, the choice of positive versus negative mode, etc. (0098. As used herein, the term “desorption” refers to 0094. As used herein, the term “mass spectrometry” or translocation of an analyte from a liquid Surface and/or the “MS refers to an analytical technique to identify compounds entry of an analyte into a gaseous phase. Laser desorption by their mass. MS refers to methods of filtering, detecting, thermal desorption is a technique wherein a sample contain and measuring ions based on their mass-to-charge ratio, or ing the analyte is thermally desorbed into the gas phase by a “m/z. MS technology generally includes (1) ionizing the laser pulse. The laser hits the back of a specially made 96-well compounds to form charged compounds; and (2) detecting plate with a metal base. The laser pulse heats the base and the the molecular weight of the charged compounds and calcu heat causes the sample to transfer into the gas phase. The gas lating a mass-to-charge ratio. The compounds may be ionized phase sample is then drawn into the mass spectrometer. and detected by any suitable means. A "mass spectrometer 0099. As used herein, the term “selective ion monitoring generally includes an ionizer and an ion detector. In general, is a detection mode for a mass spectrometric instrument in one or more molecules of interest are ionized, and the ions are which only ions within a relatively narrow mass range, typi Subsequently introduced into a mass spectrographic instru cally about one mass unit, are detected. US 2015/03461 70 A1 Dec. 3, 2015

0100. As used herein, “multiple reaction mode, some 0104. In particularly preferred embodiments, the analytes times known as “selected reaction monitoring.” is a detection of interest are quantified in a sample using MS/MS as follows. mode for a mass spectrometric instrument in which a precur One or more of the analytes of interest in samples are first sor ion and one or more fragment ions are selectively filtered through and eluted from a solid phase extraction detected. column at a high pH in the presence of FMOC-CI or a variant 0101 The ions may be detected using several detection thereof. The resulting eluent is then subjected to liquid chro modes. For example, selected ions may be detected, i.e. using matography, preferably HPLC. The flow mobile phase from a selective ion monitoring mode (SIM), or alternatively, ions the chromatographic column enters the heated ESI probe of may be detected using a scanning mode, e.g., multiple reac an MS/MS analyzer and the analytes ionized. The ions, e.g. tion monitoring (MRM) or selected reaction monitoring precursor ions, pass through the orifice of the instrument and (SRM). Preferably, the mass-to-charge ratio is determined enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) using a quadrupole analyzer. For example, in a "quadrupole' are mass filters, allowing selection of ions (i.e., selection of or "quadrupole ion trap' instrument, ions in an oscillating “precursor and “fragment” ions in Q1 and Q3, respectively) radio frequency field experience a force proportional to the based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) DC potential applied between electrodes, the amplitude of the is the collision cell, where ions are fragmented. The first RF signal, and the mass/charge ratio. The Voltage and ampli quadrupole of the mass spectrometer (Q1) selects for mol tude may be selected so that only ions having a particular ecules with the mass to charge ratios the analytes of interest. mass/charge ratio travel the length of the quadrupole, while Precursor ions with the correct mass/charge ratios are allowed all other ions are deflected. Thus, quadrupole instruments to pass into the collision chamber (Q2), while unwanted ions may act as both a “mass filter and as a “mass detector' for the with any other mass/charge ratio collide with the sides of the ions injected into the instrument. quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This 0102 One may enhance the resolution of the MS tech process is called collision activated dissociation (CAD), or nique by employing “tandem mass spectrometry,” or “MS/ collision induced dissociation (CID). The fragmentions gen MS'. In this technique, a precursor ion (also called a parent erated are passed into quadrupole 3 (Q3), where the fragment ion) generated from a molecule of interest can be filtered in an ions are selected while other ions are eliminated. During MS instrument, and the precursor ion is Subsequently frag analysis of a single sample, Q1 and/or Q3 may be adjusted mented to yield one or more fragmentions (also called daugh Such that mass/charge ratios of one or more precursor ion/ terions or productions) that are then analyzed in a second MS fragment ion pairs specific to one catecholamine is first filter and detector (quadrupole). By careful selection of pre selected, followed at some later time by the selection of cursor ions, only ions produced by certain analytes are passed mass/charge ratios of one or more precursorion/fragmention to the fragmentation chamber, where collisions with atoms of pairs specific to a second catecholamine, optionally followed an inert gas produce the fragment ions. Because both the at Some later time by the selection of mass/charge ratios of precursor and fragment ions are produced in a reproducible one or more precursor ion/fragment ion pairs specific to a fashion under a given set of ionization/fragmentation condi third catecholamine and so on. In particularly preferred tions, the MS/MS technique may provide an extremely pow embodiments, mass to charge ratios of precursor/fragment erful analytical tool. For example, the combination of filtra ion pairs specific to epinephrine, mass to charge ratios of tion/fragmentation may be used to eliminate interfering precursor/fragmention pairs specific to norepinephrine, mass Substances, and may be particularly useful in complex to charge ratios of precursor/fragment ion pairs specific to samples, such as biological samples. dopamine, mass to charge ratios of precursor/fragments of 0103) The mass spectrometer typically provides the user metanephrine, and mass to charge ratios of precursor/frag with an ion scan; that is, the relative abundance of each ion ments of normetanephrine, are detected during analysis of a with a particular mass/charge over a given range (e.g. m/z. single sample, although the sequence of detection may occur 5-1250 for API 5000) The results of an analyte assay, that is, in any order. a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. 0105. The methods may involve MS/MS performed in For example, given that sampling and analysis parameters are either positive or negative ion mode; preferably positive ion carefully controlled, the relative abundance of a given ion mode. Using standard methods well known in the art, one of may be compared to a table that converts that relative abun ordinary skill is capable of identifying one or more fragment dance to an absolute amount of the original molecule. Alter ions of a particular precursor ion of the analyte of interest that natively, molecular standards may be run with the samples, may be used for selection in quadrupole 3 (Q3). and a standard curve constructed based on ions generated 0106. In various embodiments, the analyte of interest is from those standards. Using such a standard curve, the rela Subjected to a mass spectrometry for detection and quantifi tive abundance of a given ion may be converted into an cation. A mass spectrometry technique may employ atmo absolute amount of the original molecule. In certain preferred spheric pressure chemical ionization (APCI) or electrospray embodiments, one or more internal standards may be used to ionization (ESI) to generate charged ions. The analyte of generate standard curves for calculating the quantity of the interest can present as a proton adduct or a protonated analytes of interest. Methods of generating and using Such molecular ion, i.e. M+H" in the mobile phase. The analyte standard curves are well known in the art and one of ordinary can also be shown the ammonium adduct M+NH' as a skill is capable of selecting appropriate internal standards. molecularion when abundant ammonium ion is present in the For example, an isotopically labeled catecholamine may be mobile phase, or other cation adduct when corresponding used as an internal standard; in certain preferred embodi cations are present in the mobile phase. Different adducts are ments, d6-epinephrine and/or d6-norepinephrine and/or also possible and can be recognized by the skilled artisan, and d4-dopamine may be used as internal standards. Numerous are generally shown by M+A+H", where A is the adduct. other methods for relating the amount of anion to the amount The adducts may or may not be solvated. During the ioniza of the original molecule will be well known to those of ordi tion process, the molecular ions are desorbed into the gaseous nary skill in the art. phase, and then focused into the mass spectrometer for analy US 2015/03461 70 A1 Dec. 3, 2015 14 sis and detection. See U.S. Pat. No. 6,692,971 for more infor 0111 Internal standards, such as deuterated Metaneph mation on APCI, as it is known to those of skill of the art. rines, can be applied in the methods described herein. In one 0107 MS analysis can be done with a single mass analyzer embodiment, Metanephrines-d6 is used. In another embodi Such as a single quadrupole mass spectrometer (MS), or a ment, Normetanephrines-d6 is employed. The MRM transi tandem mass analyzer Such as a triple quadrupole tandem tion pairs are listed as below. mass spectrometer (MS/MS). In a tandem mass spectrometry (O112 Molecular ions M+NH" of Fmoc derivatives of mode, the first mass filter or quadrupole (Q1) can be tuned to thyroid hormones are shown at below as MRM transitions of select independently, one or more of the molecular ions of the precursor-production pair can be monitored for thyroid hor analyte of interest and internal standards of choice. The mones are listed below as examples: molecular ions (precursor ions) can undergo collision-in duced dissociation (CID) at second quadrupole (Q2) to pro duce fragment or product ions. The fragment ions can be rT3-2FMoc NH4 T3-2FMOc NH4 T4-2FMoc NH4 detected and analyzed at the second mass filter at Q3. This process can be referred to as product optimization. The sec 1112.79 695.9 1112.9 696.1 1239.01 1791 1112.79 179.2 1112.9 179.0 1239.01 821.8 ond mass filter is then tuned to selectively monitor one or 1112.79 8743 1112.9 634.6 1239.01 2784 more of the most abundant product ions produced from a 1112.79 178.1. 1112.9 178.2 1239.01 178.0 particular molecular ion. This technique is called multiple 1112.79 2O7.O 1112.9 649.9 1239.01 1008.3 reaction monitoring (MRM). 1112.79 1029.8 1112.9 6SO.O 1239.01 708.6 0108 Molecular ions M+NH" of Fmoc derivatives of 1112.79 281.O 1112.9 517.4 1239.01 1 OO6.4 catecholamines are shown at below as MRM transitions of 1112.79 262.9 1112.9 263.O 1239.01 731.9 precursor-production pair can be monitored for catechola 1112.79 283.2 1112.9 768.3 1239.01 337.0 1112.9 SO7.8 1113 479.O mines are listed below as examples: 1113.01 177.2 1113.01 605.7 1113.01 606.1 Dopamine- Epinephrine- Norepinephrine 1113.01 855.4 3Fmoc-NH 3Fmoc-NH 3Fmoc-NH 837.411 179.1 867.19 298.4 853.239 196.2 0113 Molecular ions M+H" of Fmoc derivatives of thy 837.411 180.1 867.19 166.O 853.239 178.0 roid hormones are shown at below as MRM transitions of 837.411 178.0 867.19 179.1 853.239 152.1 837.411 137.3 867.19 178.0 853.239 179.1 precursor-production pair can be monitored for thyroid hor 837.411 91.O 867.19 210.2 853.239 107.1 mones are listed below as examples: 837.411 119.O 867.19 192.1 853.239 135.2 837.117 196.7 867.342 850.2 837.117 183.0 rT3-2FMoc H T3-2FMoc H T4-2FMoc H 837.117 261.8 837.117 198.5 1096.07 1012.7 1096.11 1012.8 1221.96 1054.7 1096.O7 928.S. 1096.11 928.6 1221.96 97O.S 1096.O7 8445 1096.11 844.S 1221.96 337.1 0109 Internal standards, such as deuterated catechola 1096.O7 SO6.7 1096.11 SO6.7 1221.96 260.9 mines, can be applied in the methods described herein. In one 1096.O7 2O6.9 1096.11 516.7 1221.96 262.9 1096.O7 291.2 1096.11 265.O 1221.96 484.6 embodiment, Dopamine-dal is used. In another embodiment, 1096.O7 263.1 1096.11 262.9 1221.96 206.9 Epinephrine-d6 is employed. Yet in another embodiment, 1096.O7 26S.O. 1096.11 2O7.1 1221.96 291.2 Norepinephrine-d6 is employed. The MRM transition pairs 1096.O7 273.O. 1096.11 2811 1221.96 1180.3 are listed as below: 1096.6 SO8.8 1096.11 479.O

Dopamine-D4 (+) 841 179 0114 Internal standards can be applied in the methods (+) 841 184 described herein. In one embodiment, 3.3',5-triiodo-L-thyro Epinephrine-D6 (+) 873 3O4 nine-'C (T3-'C) is used. The MRM transition pairs are (+) 873 179 listed as below. Norepinephrine-D6 (+) 859 290 (+) 859 2O2 3,3',5-Triiodo (+) Quantitative 1118.9 701.8 0110 Molecular ions M+NH4+ of Fmoc derivatives of L-thyronine-C (T3-'C) (+) Confirmatory 1118.9 655.9 metanephrine and Normetanephrine are shown with the MRM transitions of precursor-production pairs as examples: 0115 Molecular ions M+NH of Fmoc derivatives of THC are shown at below as MRM transitions of precursor Metanephrine- Normetanephrine production pair can be monitored for THC are listed below as 2FMOC NH3 2FMOC NH3 examples:

659.21 2684 645.1 1661 659.21 624.3 645.1 2102 THC-FMoc NH 659.21 1791 645.1 2S4.3 659.21 642.5 645.1 178.0 SS433 537.2 659.21 1801 645.1 1790 SS433 371.0 659.21 178.0 645.1 1493 SS433 1793 659.21 446.5 645.1 121.2 SS433 355.0 659.21 224.3 645.1 1340 SS433 178.1 659.21 16S.O 645.1 106.0 SS433 206.9 US 2015/03461 70 A1 Dec. 3, 2015

0116. The methods disclosed herein can be evaluated by (0123. 3. The method of embodiment 1 or embodiment 2, several parameters including, e.g., accuracy, precision, limit wherein the one or more analytes is a compound having a of detection (LOD), limits of quantitation (LOO), linear primary amine, a secondary amine, or a phenolic hydroxyl range, specificity, selectivity, linearity, ruggedness, and sys group. tem suitability. The accuracy of a method is the measure of 0.124. 4. The method of any one of embodiments 1-3, exactness of an analytical method, or the closeness of agree wherein the one or more analytes is a drug, a hormone, a ment between the measured value and the value that is signaling agent, an amino acid, or a pesticide. accepted as a conventional true value oran accepted reference 0.125 5. The method of any one of embodiments 1-4, value. The precision of a method is the degree of agreement wherein the one or more analytes is a monoamine neu among individual test results, when the procedure is applied rotransmitter including catecholamine or one of its deriva repeatedly to multiple samplings of a homogeneous sample. tives or metabolites, a sex hormone or one of its derivatives As such, precision evaluates 1) within assay variability; 2) or metabolites, a cannabinoid or one of its derivatives or within-day variability (repeatability); and 3) between-day metabolites, a thyroid hormone or one of its derivatives or variability (intermediate precision); and 4) between-lab vari metabolites, an opiate, opioid or one of its derivatives or ability (reproducibility). Coefficient of variation (CV%) is a metabolites oran arylcyclohexylamine or one of its deriva quantitative measure of precision expressed relative to the tives or metabolites, an Amphetamine or one of its deriva observed or theoretical mean value. The limit of detection tives or metabolites. (LOD) of a method refers to the concentration of analyte 0.126 6. The method of any one of embodiments 1-5, which gives rise to a signal that is significantly different from wherein the catecholamine or one of its derivatives or the negative control or blank and represents the lowest con metabolites is Catechol, Dopa, Dopamine, Epinephrine, centration of analyte that can be distinguished from back Norepinephrine, 3-Methoxytyramine, p-Octopamine, ground. Synephrine, Tyramine, 3,4-dihydroxybenzylamine, 3,4- 0117 The limits of quantitation (LOO) are the lowest and Dihydroxymandelic acid, Dihydroxyphenylethylene gly the highest concentrations of analyte in a sample that can be col, DOPAL, DOPAC, Homovanillic acid, Hydroxytyro measured with an acceptable level of accuracy and precision. Sol, 3-Methoxy-4-hydroxyphenylglycol, The lower limit of quantitation refers to the lowest dose that a 3-Methoxytyramine (3-MT), MOPET, Normetanephrine, detection method can measure consistently from the back metanephrine, or . ground. The upper limit of quantitation is the highest dose 0127 7. The method of embodiment 5, wherein monoam that a detection method can measure consistently before satu ine neurotransmitter is Histamine, Tryptamine, Serotonin, ration of the signal occurs. The linear range of the method is or Agmatine. the area between the lower and the upper limits of quantita 0.128 8. The method of embodiment 5, wherein the sex tion. The linear range is calculated by Subtracting lower limit hormone or one of its derivatives or metabolites is an of quantitation from the upper limit of quantitation. As used estrogen. herein, the term 'signal to noise ratio for the lower asymp tote' refers to the signal detected in the method at the lower 0129. 9. The method of embodiment 8, wherein the estro limit of detection divided by the background signal. As used gen is Estrone, Estradiol, Estriol, or Estetrol. herein, the term “signal to noise ratio for the upper asymp 0.130 10. The method of embodiment 5, wherein the can tote' refers to the signal detected in the method at the upper nabinoid or one of its derivatives or metabolites is a Can limit of detection divided by the background signal. nabigerol-type (CBG) cannabinoid, a Cannabichromene 0118. As used herein, an “amount of an analyte in a body type (CBC) cannabinoid, a Cannabidiol-type (CBD) fluid sample refers generally to an absolute value reflecting cannabinoid, a Cannabinodiol-type (CBND) cannabinoid, the mass of the analyte detectable in volume of body fluid. a Tetrahydrocannabinol-type (THC) cannabinoid, a Can However, an amount also contemplates a relative amount in nabinol-type (CBN) cannabinoid, a Cannabitriol-type comparison to another analyte amount. For example, an (CBT) cannabinoid, a Cannabielsoin-type (CBE) cannab amount of analyte in a body fluid can be an amount which is inoid, an Isocannabinoid, a Cannabicyclol-type (CBL) greater than a control or normal level of analyte normally cannabinoid, a Cannabicitran-type (CBT) cannabinoid, or present. a Cannabichromanone-type (CBCN) cannabinoid. I0131 11. The method of embodiment 5, wherein the thy 0119 The present methods and kits may be used for the roid hormone or one of its derivatives or metabolites is 3,3', quantification or the detection of an analyte of interest. 5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), or 0120 Aspects of the present specification may also be thyroxine (T4). described as follows: I0132) 12. The method of embodiment 5, wherein the opi 0121 1. A method for determining the presence one or ate, the opioid or the derivative or metabolite of the opiate more analytes in a test sample, the method comprising: a) or opioid, is morphine, oripavine, morphinone, hydromor Solid phase extraction of the one or more analytes from the phone, or oxymorphone. test sample, b) elution of the one or more analytes from the 0.133 13. The method of embodiment 5, wherein the opi Solid phase extraction with an elution Solution having a ate, the opioid, or the derivative or metabolite of the opiate basic pH, or an acidic pH or a neutral pH c) in situ deriva or opioidisabenzylisoquinoline alkaloid, a semi-synthetic tization of the one or more analytes with a derivatizing benzylisoquinoline alkaloid derivative, or an opioid. agent, and d) detecting the one or more derivatized analytes 0.134 14. The method of embodiment 5, wherein the aryl using liquid chromatography and/or mass spectrometry. cyclohexylamine or one of its derivatives or metabolites is 0122 2. A method for optimizing solid phase extraction Tiletamine, 3-Methoxetamine (MXE), Methoxyketamine, (SPE) protocols of one or more analytes in a biological N-Ethylnorletamine (Ethiketamine), Amphetamine, Ephe sample via in situ derivatization and LC-MS/MS detection. drine, or Methamphetamine. US 2015/03461 70 A1 Dec. 3, 2015

0135 15. The method of embodiment 5, wherein the 0151. 31. The method of embodiment 30, wherein R', R, Amphetamine or one of its derivatives or metabolites is R, and R are each independently, H, fluorine, chlorine, Amphetamine (itself), methamphetamine, ephedrine, bromine, iodine, a cyano group, an acetylene group, a cathinone, 3.4-methylenedioxy-N-methylamphetamine propylene group, a vinyl group, a linear, branched, or (MDMA, "Ecstasy”), and 2,5-Dimethoxy-4-methylam cyclic Cls alkyl, a Substituted vinyl group, or a linear or phetamine (DOM, or "STP"). branched or cyclic Cls alkoxy group. 0136. 16. The method of any one of embodiments 1 or 3-15, wherein the solid phase extraction is performed with 0152 32. The method of embodiment 31, wherein the an ion exchange column or cartridge. FMOC compound is shown in Formula III: 0137) 17. The method of embodiment 16, wherein the ion (III) exchange column is a cation exchange column. R 0.138. 18. The method of embodiment 17, wherein the cation exchange column is a weak cation exchange col l 0139 19. The method of embodiment 17, wherein the cation exchange column is a strong cation exchange col l 0140. 20. The method of embodiment 16, wherein the ion exchange column is an anion exchange column. 0141. 21. The method of any one of embodiments 1 or ( ) 3-20, wherein the solid phase extraction is performed with a reverse phase silica column or cartridge. 0142 22. The method of embodiment 21, wherein the 0153 wherein R' and R are each independently, H, reverse phase silica is an alkyl bounded (C4, C8, C12, or fluorine, chlorine, bromine, iodine, a cyano group, an C18) silica, a cyano bounded silica, a phenyl bounded acetylene group, a propylene group, a Vinyl group, a silica, or a biphenyl bounded silica. linear, branched, or cyclic Cls alkyl, a Substituted vinyl 0143. 23. The method of one any of embodiments 1-22, wherein the sample is a biological sample, a Soil sample, or group, or a linear or branched or cyclic Cls alkoxy a sample of food stuff. group. 0144. 24. The method of embodiment 23, wherein the 0154 32. The method of embodiment 31, wherein the biological sample is a blood sample, a saliva sample, a Fmoc chloride is represented as follows: lachrymal sample, a urine sample, or a tissue sample. (0145 25. The method of embodiment 24, wherein the blood sample is a full blood sample, a plasma sample, or a serum sample. 0146 26. The method of any one of embodiments 1 or 3-25, wherein the basic pH is a pH of greater than about 8. 0147 27. The method of embodiment 26, wherein the basic pH is a pH in the range of about 8 to about 13. 0148 28. The method of any one of embodiments 1-27, ( ) wherein the derivatizing reagent is an acylating agent of Formula I: (I) --, Or) 1.O 0149 29. The method of embodiment 28, wherein the acylating agent is an acyl chloride or acyl halide. 0150. 30. The method of embodiment 29, wherein the acyl chloride is Fmoc chloride Formula II.

(II) Br O-l. ( ) Br US 2015/03461 70 A1 Dec. 3, 2015 17

(O155 33. The method of embodiment 29, wherein the acyl (0160 37. The method of embodiment 29, wherein the chloride is Fmoc chloride Formula VII: acylation reagent is: (VII) (VIII) R1 O

R2 O ls C

R3 R5 R4 (0161 wherein R', R. R. R', and Rare each indepen 0156 34. The method of embodiment 33, wherein R', R, dently H, a halogenatom, optionally, fluorine, or a chlo R. R. and Rare each independently H, a halogen atom rine, or a bromine, or an iodine, a cyano group, an Such as a fluorine, or a chlorine, or a bromine, or an iodine, acetylene group, a propylene group, a Vinyl group, a a cyano group, an acetylene group, a propylene group, a linear or branched Cls alkyl, or members of a cyclic vinyl group, a linear or branched, or members of a cyclic Cs alkyl, a Substituted vinyl group, a linear or branched Cs alkyl, a Substituted vinyl group, or a linear or branched Cs alkoxy group, or members of a cyclic Cls alkoxy Cs alkoxy group or members of a cyclic Cls alkoxy group. group. (0162 39. The method of embodiment 37, wherein For O157 35. The method of embodiment 33, whereinformula mula VIII may be represented as: VII is represented as: RI O RI O O R2 R2 C or C R2 O ls C or R3 R7 R3 0.163 wherein the fused ring system may be an all car R O bocyclic or a heterocyclic aromatic system, having a 5-7 R2 ls membered fused ring, or a nonaromatic carbocyclic, or O C heterocyclic fused ring system Such as 3,4-dihydro-1H indene, or a 3.4.dihydro-2H-chromene, or having a 4-8 R7 membered fused ring, and wherein the fused ring may be substituted when appropriate with one or more of a halogen atom, a cyano group, an acetylene group, a propylene group, a vintyl group, a linear, branche or 0158 wherein the fused ring system is an all carbocy cyclic Cls alkyl, a substituted vinyl group, or a linear or clic or a heterocyclic aromatic system, optionally a branched or cyclic Cls alkoxy group. naphthalene, a quinolone, an isoquinoline, a quinzoline, (0164 40. The method of embodiment 37, wherein For a benzofuran, an indole, or a benzimidazole, with a 5-7 mula VIII is benzoyl chloride: membered substituted or unsubstituted fused ring, or a nonaromatic carbocyclic, or heterocyclic fused ring sys O tem such as 3,4-dihydro-1H-indene, or a 3.4.dihydro 2H-chromene, with a 4-8 membered fused ring, wherein C. the fused ring may be substituted when appropriate by one or more of a halogen atoms, optionally fluorine, chlorine, bromine, or iodine, a cyano group, an acety lene group, a propylene group, a Vinyl group, a linear, (0165 41. The method of embodiment 29, wherein the branched, or cyclic Cls alkyl, a substituted vinyl group, acylating reagent is Formula IX: or a linear, branched, or cyclic Cls alkoxy group. (IX) 0159) 36. The method of embodiment 33, wherein For RI O O mula VII is 2 V/ R Sn C O ls R3 R5 O C. R4 (0166 wherein R. R. R. R', and Rare each indepen dently, a hydrogen, a halogen atom, optionally fluorine, US 2015/03461 70 A1 Dec. 3, 2015

chlorine, bromine, or iodine, a cyano group, an acety 0172 47. The method of embodiment 46, wherein the lene group, a propylene group, a Vinyl group, a linear, LC-MS/MS techniques comprise Atmospheric Pressure branched, or cyclic Cls alkyl, a Substituted vinyl group, Chemical Ionization (APCI), or Electrospray Ionization or a linear or branched or cyclic Cls alkoxy group. (ESI) technique. (0167 43. The method of embodiment 41, wherein For (0173 48. The method of embodiment 46, wherein the mula IX is: LC-MS/MS techniques comprise the use of a triple qua drupole mass spectrometer instrument in Multiple Reac tion Monitoring (MRM), or Selected Reaction Monitoring (SRM), positive-ion mode. 0.174 49. The method of embodiment 48, wherein the LC-MS/MS techniques comprise a Q1 scan tuned to select a precursor ion that corresponds to the M+H, or M+NH, or M+A+H" of the acylated derivatives of the O desired one or more analytes for product optimization, wherein A is a molecular adduct, Such as an acetonitrile, or 0168 wherein the fused ring system may be an all car an H.O. bocyclic or a heterocyclic aromatic system, optionally a (0175 50. The method of embodiment 49, wherein the one naphthalene, a quinolone, an isoquinoline, a quinzoline, or more analytes are at least one of Dopamine, Epineph a benzofuran, an indole, or a benzimidazole, having a rine, and Norepinephrine, the acylated derivatives of the 5-7 membered fused ring, or a nonaromatic carbocyclic, one or more analytes are tri-Fmoc Dopamine (IM+H or heterocyclic fused ring system optionally 3,4-dihy =820, or M+NH=837), tri-Fmoc Epinephrine (IM+H dro-1H-indene, 3.4.dihydro-2H-chromene, having a 4-8 =850, or M+NH=867), and tri-Fmoc Norepinephrine membered fused ring, and wherein the fused ring may be (M+H=836, or M+NH=853). Substituted when appropriate by one or more of a halo (0176 51. The method of embodiment 49, wherein the one gen atoms, optionally fluorine, chlorine, bromine, or more analytes are at least one of Metanephrine, and iodine, a cyano group, an acetylene group, a propylene Normetanephrine and the acylated derivatives of the one or group, a Vinyl group, a linear, branched, or cyclic Cls more analytes are bis-Fmoc Metanephrine (M--H=642, alkyl, a Substituted vinyl group, or a linear or branched or M+NH=659), and bis-Fmoc Normetanephrine (M- or cyclic Cls alkoxy group. H=628, or M+NH=645). (0169. 44. The method of embodiment 28, wherein X is: C1, 0177 52. The method of embodiment 49, wherein the one or more analytes are at least one of Thyroxine (T4), and Br, F, 3.3',5-triiodothyroine (T3), and 3,3',5'-triiodothyronine (rT3), the acylated derivatives of the one or more analytes are bis-Fmoc T4 M+H=1222, or M+NH=1239), bis N2 Fmoc T3 (M+H=1096, or M+NH=1113), and bis 2 N, s N / \ s Fmoc rT3 (M+H=1096, or M+NH=1113). N f o1 o1 NF 0.178 53. The method according to embodiment 49, F wherein the one or more analytes are at least one of 8-9- O tetrahydrocannabinol (THC) and 11-nor-9-carboxyl-d-9- F F tetrahydrocannabinol (9-carboxyl-THC) and the acylated NO2, s N s derivative of 6-9-tetrahydrocannabinol (THC) is Fmoc e O F O THC (M+H=537, or M+NH=554), and the acylated derivative of 11-nor-9-carboxyl-d-9-tetrahydrocannabinol O F O (9-carboxyl-THC) is Fmoc-9-carboxyl-THC (IM+H O SONa O =567, M+NH=584). (0179 54. The method of any one of embodiments 1-53, o-N O-N wherein the detection of the analyte is qualitative or quan titative.

O O O 0180 55. A method for determining the amount of one or more one or more analytes in a sample the method com prising: elution of at least one of the one or more analytes 0170 45. The method of any one of embodiments 1-44, from a solid phase extraction with an elution buffer com wherein the mass spectrometry comprises tandem mass prising a derivatization agent under conditions that allow spectrometry techniques the derivatization the at least one of the one or more ana 0171 46. The method of any one of embodiments 1-45, lytes. wherein the mass spectrometry comprises LC-MS/MS 0181 56. A solid phase extraction kit for determining the techniques. amount of one or more analytes, the kit comprising: a) a US 2015/03461 70 A1 Dec. 3, 2015 19

Solid phase extraction column, and b) an elution Solution 0193 The sample was eluted from the column with 0.5 ml having a high pH with a derivatizing reagent in the same or of Elution Buffer; a 4% w/v FMoc-CI in 25 mMKCO, solute a separate container. in a solution of water and acetonitrile. (H2O: Acetonitrile=1: 0182 57. The kit of embodiment 56, wherein the deriva 3). The derivatization reaction was permitted to proceed for tization agent is an acylating reagent. 12 minutes at room temperature. The derivatization reaction 0183) 58. The kitofembodiment 57, wherein the acylating was stopped with 20LL of 10 mMammonium formate. 25uL agent is an acyl chloride or acyl halide. of the solution obtained from the ammonium formate reaction 018.4 59. The kit of embodiment 58, wherein the acyl was used directly as the sample injected in the LC-MS/MS chloride is an FMOC-CI or a variant thereof. analysis. 0185. 60. The kit of embodiment 58, wherein the acyl (0194 For LC-MS/MS analysis, 25 uL of the solution chloride is a CBZ-CI or a variant thereof. obtained from the ammonium formate reaction was automati 0186 61. The kit of any one of embodiments 56-60, cally injected into a TARGAR C183 um particle size 50x2.1 wherein the Solid phase extraction column is a weak cation mm analytical column. A binary HPLC gradient was applied exchange column. to the analytical column to separate the FMoc derivatives of 0187 62. The kit of any one of embodiments 56-60, epinephrine, norepinephrine and dopamine from other ana wherein the Solid phase extraction column is a strong cat lytes contained in the sample. Mobile phase A was 5.0 mM ion exchange column. ammonium formate with 0.1% formic acid pH 3.0 and mobile 0188 63. The kit of any one of embodiments 56-60, wherein the Solid phase extraction column is a reverse phase B was Acetonitrile with 0.1% formic acid. The HPLC phase column. gradient proceeded at a temperature of 35°C. with a flow rate (0189 64. The kit of any one of embodiments 56-60, of 500 ul/min over five minutes as follows: 0.01 min, 50% B: wherein the Solid phase extraction column is an anion 3.0 min, 100% B, 4.0 min 100% B, 4.5 min, 50% B, 5.0 min, exchange column. 50% B. (0195 MS/MS was performed using an API 5000 triple EXAMPLES quadrupole mass spectrometer controlled by Analyst Soft 0190. The following non-limiting examples are provided ware Version 1.52 (ABI-SCIEX, Toronto, Canada). Analyte for illustrative purposes only in order to facilitate a more exiting the HPLC analytical column through the mobile complete understanding of the disclosed Subject matter. phase flowed to the heated nebulizer interface of the MS/MS These examples should not be construed to limit any of the analyzer. The solvent/analyte mixture was converted to vapor embodiments described in the present specification, includ in the heated tubing of the interface. Analytes in the nebulized ing those pertaining to the methods for detecting an analyte solvent were ionized by heated Electrospray Ionization and kits comprising the components necessary to perform the SOUC. disclosed methods. 0.196 Ions passed to the first quadrupole (Q1), which selected ions with a mass to charge ratio of parent ions gen Example 1 erated from one of the analytes. Ions entering quadrupole 2 (Q2) collided with collision gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. Quantification of Catecholamines in Blood Plasma Simultaneously, the same process using isotope dilution mass 0191 Plasma samples were obtained from human spectrometry was carried out with internal standards, dopam patients’ blood. Samples were drawn (plasma Sodium heparin ine-D4 and/or epinephrine-D6, and/or norepinephrine-D6. & EDTA) into pre-chilled Vacutainers. Vacutainers were The following mass transitions were used for detection and inverted 5x and refrigerated until centrifuged. Plasma was quantitation of epinephrine, norepinephrine and dopamine separated in a refrigerated centrifuge (1000xg for 10 minutes) (and their corresponding internal standards) during validation within 30 minutes of collection and then frozen immediately on positive polarity from the same sample injection. at -20°C. in plastic vials. Plasma was thawed and diluted before use in solid phase extraction. The blanks, calibration TABLE 1 samples, and plasma samples were spiked with internal stan Ions Monitored dards (e.g., dopamine-D4, epinephrine-D6, and norepineph rine-D6). Standard curves were generated with plasma solu Compound Polarity Precursor miz Product m2 tions spiked with a known amount of catecholamine. The Dopamine (+) 837 18O (+) 837 137 spiking solution was serially diluted before being added to the Dopamine-D4 (+) 841 179 plasma taken from the same plasma sample. (+) 841 184 Epinephrine (+) 867 298 (0192 CEREX(R) PWCX HP (1 cc/10 mg) columns (cata (+) 867 166 log number 675-0101R) were conditioned with 0.5 ml of Epinephrine-D6 (+) 873 3O4 methanol, followed by 0.5 ml of 10 mM Phosphate Buffer pH (+) 873 179 Norepinephrine (+) 853 196 6.8. Another 0.5 ml of 10 mM phosphate buffer at pH 6.8 was (+) 853 152 added to the column. Then 0.5 ml 10 mM Phosphate buffer Norepinephrine-D6 (+) 859 290 was mixed with 100LL of the Sample. The Sample/buffer mix (+) 859 2O2 was loaded onto the column at a pressure of 2-3 psi. The column was washed with 1 ml deionized water at 2-3 psi and 0.197 The structure of the derivatized catecholamines is subsequently washed with 1 ml Acetonitrile at 6 psi. provided below (the ammonium adducts). US 2015/03461 70 A1 Dec. 3, 2015 20

O) r O

HO N HO ot CH' O HO HO Tri-Fmoc-Dopamine ammonium adduct Chemical Formula: CoHoNO3." Chemical Formula: C5H45N2Os" Chemical Formula: C6H6O2" Exact Mass: 180.07 Exact Mass: 837.32 Exact Mass: 137.06

D O D H O N O D O D O O

O)=0 NH --

D D H C O CH' HO N n C+

D D O HO Tri-Fmoc-Dopamine-da-ammonium adduct Chemical Formula: CoHo D4NO3" Chemical Formula: C5H41D4N2Os" Chemical Formula: C14H11" Exact Mass: 184.09 Exact Mass: 841.34 Exact Mass: 179.09 US 2015/03461 70 A1 Dec. 3, 2015 21

-continued

OH

OH C N NH' HO HO

HO O Tri-Fmoc-Norepinephrinei HO ammonium adduct Chemical Formula: CoHoNO" Chemical Formula: Cs3H45N2Oo" Chemical Formula: CsHoNO" Exact Mass: 196.06 Exact Mass: 853.31 Exact Mass: 152.07

Tri-Fmoc-Norepinephrine-d6 ammonium adduct Chemical Formula: CH4D6NOs" Chemical Formula: C5H3oD6N2O)" Chemical Formula: CoHo DoNO4" Exact Mass: 290.08 Exact Mass: 859.35 Exact Mass: 202.10 US 2015/03461 70 A1 Dec. 3, 2015 22

-continued

OH

OH 'N, Y- OH C) / O O N NH' HO X- O Tri-Fmoc-Epinephrine HO HO ammonium adduct Chemical Formula: C12H12NOs" Chemical Formula: C54H47NOo" Chemical Formula: CoH12NO" Exact Mass: 298.06 Exact Mass: 867.33 Exact Mass: 166.09

D O

C) C-t

Tri-Fmoc-Epinephrine-d6 ammonium adduct Chemical Formula: C12H6D6NOs" Chemical Formula: C54H2D6N2O)" Chemical Formula: C14H11" Exact Mass: 304.09 Exact Mass: 873.37 Exact Mass: 179.09

0198 Table 2 shows the data representing the percent data showing the chromatograms for the internal standard recovery of catecholamine from a plasma sample spiked with equivalent (FIG. 1A) and for the catecholamine spiked 1 ng/ml catecholamine. FIGS. 1A and 1B present example plasma (FIG. 1B). TABLE 2 Absolute Recovery: 1 ng/ml in Plasma Analyte IS Peak Mean % C.V. Peak Area Area (Area SD (Area (Area % Sample Analyte (counts) (counts) Area. Ratio Ratio) Ratio) Ratio) Recovery Std. 1 DA-1 436OOO 416OOO 1.OS 1.06 O.O404 3.8 Std. 2 114OOO 112OOO 1.02 Std. 3 378OOO 342OOO 1.1 Elution 1 668000 498OOO 1.34 1.26 O.O883 7.0 119.2 Elution 2 636OOO SS8000 1.14 Elution 3 696OOO SS8000 1.25

US 2015/03461 70 A1 Dec. 3, 2015 24

TABLE 3-continued was added to the column. The Sample/buffer mix was loaded onto the column at a pressure of 2-3 psi. The column was Linearity - 10 pg/ml to 100 ng/ml in Plasma washed with 1 ml deionized water at 2-3 psi and subsequently washed with 1 ml Acetonitrile at 6 psi. The sample was eluted Analyte Analyte S Peak Calculated Accu from the column with 0.5 ml of Elution Buffer; 20 ul of 100 Analyte Conc. Peak Area Area Area Conc. racy mg/ml FMOC-CI in a 50:50 solution of 100 mM K2CO3: Peak (pg/mL) (counts) Ratio (counts) (pg/mL) (%) acetonitrile. The derivatization reaction was permitted to pro EP-2 1OOOOO 111OOOOO 87.3 27OOO 105OOO 05 ceed for 12 minutes at room temperature. The derivatization NE-1 10 993 O.OOS67 17SOOO 942 94.2 reaction was stopped with 20 uL of 20:8050mMNH4CO3H: NE-1 10 955 0.00555 172000 9.18 91.8 NE-1 1OO 916O O.OS13 78OOO 98.8 98.8 Acetonitrile. 25uL of the solution obtained from the ammo NE-1 1OO 106OO O.O476 223OOO 91.6 91.6 nium formate reaction was used directly as the sample NE-1 1OOO 91800 0.629 46000 1230 23 injected in the LC-MS/MS analysis. NE-1 1OOO 827OO O.S26 57OOO 1030 O3 (0204 For LC-MS/MS analysis, 25 uL of the solution NE-1 1OOOO 722OOO 4.9 47OOO 97.90 97.9 obtained from the ammonium formate reaction was automati NE-1 1OOOO 76SOOO 4.96 S4OOO 992O 99.2 cally injected into a TARGAR C183 um particle size 50x2.1 NE-1 1OOOOO 47SOOOO 37.5 26OOO 903OO 90.3 mm analytical column. A binary HPLC gradient was applied NE-1 1OOOOO 453OOOO 43.5 O4OOO 11 OOOO 10 to the analytical column to separate the metanephrines from NE-2 10 1O3O O.OOS86 17SOOO 9.55 95.5 NE-2 10 864 O.OOSO2 172OOO 7.47 74.7 other analytes contained in the sample. Mobile phase A was NE-2 1OO 7S4O O.O422 78OOO 99.3 99.3 5.0 mM ammonium formate with 0.1% formic acid pH 3.0 NE-2 1OO 962O O.O432 223OOO 102 O2 and mobile phase B was Acetonitrile with 0.1% formic acid. NE-2 1OOO 70300 0.482 46000 1190 19 The HPLC gradient proceeded at a temperature of 35°C. with NE-2 1OOO 72700 0.462 57OOO 1140 14 a flow rate of 500 ul/min over five minutes as follows: 0.01 NE-2 1OOOO 572OOO 3.88 47OOO 97.30 97.3 min, 50% B;3.0 min, 100% B, 4.0 min 100% B, 4.5 min, 50% NE-2 1OOOO 609000 3.95 S4OOO 9900 99 B, 5.0 min, 50% B. NE-2 1OOOOO 422OOOO 33.3 26OOO 976OO 97.6 (0205 MS/MS was performed using an API 5000 triple NE-2 1OOOOO 361OOOO 34.7 O4OOO 102OOO O2 quadrupole mass spectrometer controlled by Analyst Soft ware Version 1.52 (ABI-SCIEX, Toronto, Canada). Analyte 0201 FIGS. 2A and 2B represent data obtained at 10 and exiting the HPLC analytical column through the mobile 100 pg/ml. However, all concentrations were read, with two phase flowed to the heated nebulizer interface of the MS/MS plasma samples each, and each sample being read in dupli analyzer. The solvent/analyte mixture was converted to vapor cate. The data summarized in Table 4 provide exemplary in the heated tubing of the interface. Analytes in the nebulized linearity regression data for the quantification of dopamine, solvent were ionized by heated Electrospray Ionization Epinephrine and Norepinephrine in plasma. SOUC. TABLE 4

Extraction Analyte Efficiency (%)" LLOQ Linear Range Mean C.V. Mean R2 Dopamine 1113 10 pg/ml 10 pg/ml-100 ng/ml 7.2 O.9983 Epinephrine 99 10 pg/ml 10 pg/ml-100 ng/ml 10.2 O.9989 Norepinephrine 82.5 10 pg/ml 10 pg/ml-100 ng/ml 8.8 O.9985 Average Extraction Efficiency of Dopamine 837 > 180 & 837 > 137, Epinephrine 867 > 298 & 867 > 166, & Norepinephrine 853 > 196 & 853 > 152 °C.V. of Triplicates from Recovery Data (average for both ions) Quadratic Regression (1ix weighing) of Standard Curve

Example 2 0206 Ions passed to the first quadrupole (Q1), which selected ions with a mass to charge ratio of parent ions gen Quantification of Metanephrines in Blood Plasma erated from one of the analytes. Ions entering quadrupole 2 Metanephrines Method and Example Data (Q2) collided with argon gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. After 0202 Plasma samples were obtained from human measurement of ions indicative of one of the analytes, Q1 was patients’ blood. Samples were drawn (plasma Sodium heparin adjusted so that ions with a mass to charge ratio of parention & EDTA) into pre-chilled Vacutainers. Vacutainers were from a second analyte were selected. These ions were col inverted 5x and refrigerated until centrifuged. Plasma was lided with argon gas in Q2, and the ion fragments passed to separated in a refrigerated centrifuge (1000xg for 10 minutes) Q3 for further selection. The following mass transitions were within 30 minutes of collection and then frozen immediately used for detection and quantitation of the metanephrines dur at -20°C. in plastic vials. Plasma was thawed and diluted ing validation on positive polarity from the same sample before use in solid phase extraction. The blanks, calibration injection. samples, and plasma samples were spiked with internal stan dards. Standard curves were generated with plasma Solutions spiked with a known amount of metanephrine. The spiking TABLE 5 solution was serially diluted before being added to the plasma Ions Monitored taken from the same plasma sample. 0203 CEREX(R) PWCX, 1 cc 10 mg,96/pk (catalog num Compound Polarity Precursor miz Product m2 ber 675-0101R) were conditioned with 0.5 ml of methanol, Metanephrine (+) 659.2* 268.4 followed by 0.5 ml of 10 mM Phosphate Buffer pH 6.8.0.5 ml (+) 659.2 624.3 10 mM Phosphate buffer was mixed with 100 uL of the (+) 659.2 179.1 Sample. Another 0.5 ml 10 mM Phosphate Buffer at pH 6.8 US 2015/03461 70 A1 Dec. 3, 2015 25

TABLE 5-continued (0209 CEREX(R) PSCX 1cc 10 mg (catalog number 687 0101R, 96/pk) columns were conditioned with 1.0 mL of Ions Monitored methanol, followed by 1.0 mL of 2% formic acid (aqueous). Compound Polarity Precursor miz Product miz 0.5 mL of 13% acetonitrile in 2% Formic Acid (aq) was (+) 659.2 642.5 mixed with 200LL of the Sample. 0.5 mL of 13% acetonitrile (+) 659.2 180.1 in 2%. Formic Acid was added to the column. The Sample/ (+) 659.2 178 (+) 659.2* 446.5* buffer mix was loaded onto the column at a pressure of 2-3 (+) 659.2 224.3 psi. The column was washed with 1 ml 50% acetonitrile (+) 659.2 16S Normetanephrine (+) 645.1% 166.18 (aqueous) at 3 psi and Subsequently washed with 1 ml Aceto (+) 645.1 210.2 nitrile at 6 psi. Columns were dried for 30 seconds with a (+) 645.1% 2543: stream of nitrogen at high pressure. The columns were (+) 645.1 178 (+) 645.1 179 washed with 1 ml 10 mM potassium carbonate pH 11.9, at 3 (+) 645.1 149.3 pS1. (+) 645.1 121.2 (+) 645.1 134 0210. The sample was eluted from the column with 0.5 ml (+) 645.1 106 of elution buffer 100 mM potassium carbonate (aq., pH 11.9): Acetonitrile, 20 uL of 100 mg/ml FMOC-CI in aceto nitrile was added postelution. The derivatization reaction was TABLE 6 permitted to proceed for 25 minutes at room temperature. The Recovery Data derivatization reaction was stopped with 20 uL of 20:80 50 Analyte Peak mMNHCOH (aq.): Acetonitrile. This reaction mixture was Sample Analyte Area (counts) % Recovery used directly for quantification without further treatment Standard Metanephrine 186OOOO 0211 For LC-MS/MS analysis, 20 uL of the solution of Elution - Rep 1 659 - 268 18SOOOO 99.5 Elution - Rep 2 17OOOOO 91.4 the derivatization reaction mixture was injected into a Standard Metanephrine 137OOOO TARGAR) C183 um particle size 50x2.1 mm analytical col Elution - Rep 1 659 - 446 1190OOO 86.9 Elution - Rep 2 113 OOOO 82.5 umn. A binary HPLC gradient was applied to the analytical Standard Normetanephrine 158OOOO column to separate the thyroid hormones from other analytes 645 - 166 Elution - Rep 1 152OOOO 96.2 contained in the sample. Mobile phase A was 20.0 mM Elution - Rep 2 1570000 99.4 ammonium formate pH 3.0 and mobile phase B was Aceto Standard - Rep 1 Normetanephrine 13OOOOO nitrile with 0.1% formic acid. The HPLC gradient proceeded 645 - 254 Standard - Rep 2 138OOOO 106.2 at a temperature of 40°C. with a flow rate of 500 ul/min over Standard - Rep 3 14SOOOO 111.5 five minutes as follows: 0.01 min, 60% B; 7.5 min, 85% B, 7.6 min, 95% B; 7.9 min, 95% B, 8.0 min, 60% B: 9.0 min, 60% 0207 Again, the percent recovery was very high, as shown B. by Table 6. FIGS. 4A-4D show the spectra for the metaneph 0212 MS/MS was performed using an API 5000 triple rines. quadrupole mass spectrometer controlled by Analyst Soft Example 3 ware Version 1.52 (ABI-SCIEX, Toronto, Canada). Analyte Quantification of Thyroid hormones in Blood Plasma exiting the HPLC analytical column through the mobile Thyroid Hormones (T3, rT3, T4) Method and phase flowed to the heated nebulizer interface of the MS/MS Example Data analyzer. The solvent/analyte mixture was converted to vapor 0208 Sample preparation was done as in Example 1, in the heated tubing of the interface. Analytes in the nebulized except that samples were spiked with thyroid hormones and solvent were ionized by heated Electrospray Ionization appropriate internal standards. SOUC.

I O I O

HO I O I

OH -e- OH NH2 Fmoc1 O O I I HNN Fmoc

I I

Thyroxine, T4 Bishimoc thyroxine T. (Fmoc) Chemical Formula: C6H13I4NO3 Chemical Formula: C46H33I4NO-7 Exact Mass: 774.71 Exact Mass: 1218:84 US 2015/03461 70 A1 Dec. 3, 2015 26

-continued

I O

O O I OH

O HN O I O

I O NH

Chemical Formula: C45H35I-N2Os" Exact Mass: 1238.86 Bismoc thyroxine T4 (Fmoc)2 ammonium adduct

I O

HO I OH

HN O O rN-N-N- NY I OH Chemical Formula: C14H11" Chemical Formula: C6H11NO6 Exact Mass: 179.09 Exact Mass: 820.68 C roor. O

Chemical Formula: Cash 36.13N2Os" Exact Mass: 1112.96 Bishimoc 3,5,3'-triiodothyronine T3(Fmoc) ammonium adduct I O I O

HO I HO I OH OH HN OH NH2 O Y O I OH I

Chemical Formula: C6H13INO6" Chemical Formula: C15H12INO4 Exact Mass: 695.79 Exact Mass: 650.79

0213 Ions passed to the first quadrupole (Q1), which from a second analyte were selected. These ions were col selected ions with a mass to charge ratio of parent ions gen lided with argon gas in Q2, and the ion fragments passed to erated from one of the analytes. Ions entering quadrupole 2 (Q2) collided with argon gas to generate ion fragments, which Q3 for further selection. The following mass transitions were were passed to quadrupole 3 (Q3) for further selection. After used for detection and quantitation of the thyroid hormones measurement of ions indicative of one of the analytes, Q1 was during validation on positive polarity from the same sample adjusted so that ions with a mass to charge ratio of parention injection. US 2015/03461 70 A1 Dec. 3, 2015

TABLE 7 TABLE 7-continued

Ions Monitored Ions Monitored Precursor Product Precursor Product Compound Polarity mz mz Compound Polarity mz mz Reverse (+) Quantitative 1113 696 Triiodothyronine (+) Quantitative 1113 696 triiodothyronine (rT3) (+) Confirmatory 1113 649.6 1113 649.6 3,3',5-Triiodo-L (+) Quantitative 1118.9 701.8 (T3) (+) Confirmatory -thyronine- (+) Confirmatory 1118.9 655.9 Thyroxine (T4) (+) Quantitative 1239 821 13C (T3-'C) (+) Confirmatory 1239 179.1

TABLE 8 Absolute Recovery: 5 mg ml in Plasma & BSA Analyte IS Peak Mean SD % C.V. Peak Area Area Area (Area (Area (Area % Sample Analyte (counts) (counts) Ratio Ratio) Ratio) Ratio) Recovery Standard - Rep 1 rT3 239OOO 314OOO O.761 O.726 O.O317 4.4 Standard - Rep 2 316OOO 454OOO O.697 Standard - Rep 3 376OOO SO)6OOO 0.744 Standard - Rep 4 344OOO 491 OOO O.701 Blank Plasma - Rep 1 59SO 490000 OO121 O.O13 O.OOO7 5.7 Blank Plasma - Rep 2 5240 4O2(OOO O.O131 Blank Plasma - Rep 3 SO4O 43OOOO O.O117 Blank Plasma - Rep 4 S7SO 44OOOO O.O131 Spiked Plasma - Rep 1 381OOO 48OOOO O.794 O.839 O.O334 4.0 113.8 Spiked Plasma - Rep 2 361OOO 434OOO 0.832 Spiked Plasma - Rep 3 379000 437OOO O.867 Spiked Plasma - Rep 4 374OOO 434OOO O.861 Spiked BSA - Rep 1 396OOO S62OOO O.7OS O.648 0.0473 7.3 89.3 Spiked BSA - Rep 2 341 OOO S36OOO O.63S Spiked BSA - Rep 3 318OOO S37OOO O.S92 Spiked BSA - Rep 4 343OOO S19 OOO O.661 Standard - Rep 1 T3 209 OOO 314OOO 0.665 0.779 0.0759 9.8 Standard - Rep 2 367OOO 454OOO O.808 Standard - Rep 3 416OOO SO6OOO O.822 Standard - Rep 4 4O2(OOO 491OOO O.819 Blank Plasma - Rep 1 S1600 490OOO O. 105 O.107 O.OO40 3.7 Blank Plasma - Rep 2 42300 4O2(OOO 0.105 Blank Plasma - Rep 3 48SOO 43OOOO O. 113 Blank Plasma - Rep 4 464OO 44OOOO O. 105 Spiked Plasma - Rep 1 38OOOO 48OOOO O.791 O.816 0.0261 3.2 91.0 Spiked Plasma - Rep 2 37OOOO 434OOO 0.852 Spiked Plasma - Rep 3 3S2OOO 437OOO O.805 Spiked Plasma - Rep 4 353OOO 434OOO O.814 Spiked BSA - Rep 1 397OOO S62OOO O.7O6 O.716 O.O198 2.8 91.9 Spiked BSA - Rep 2 399OOO S36OOO O.744 Spiked BSA - Rep 3 37SOOO S37OOO O.699 Spiked BSA - Rep 4 37OOOO S19 OOO O.713 Standard - Rep 1 T4 712OO 314OOO O.227 O.241 O.O141 5.8 Standard - Rep 2 109 OOO 454OOO 0.24 Standard - Rep 3 119 OOO SO)6OOO O.23S Standard - Rep 4 127OOO 491 OOO O.26 Blank Plasma - Rep 1 1090OOO 490OOO 2.23 2.343 0.1422 6.1 Blank Plasma - Rep 2 1O3OOOO 4O2(OOO 2.SS Blank Plasma - Rep 3 990OOO 43OOOO 2.31 Blank Plasma - Rep 4 1OOOOOO 44OOOO 2.28 Spiked Plasma - Rep 1 1190OOO 48OOOO 2.48 2.640 0.1826 6.9 123.7 Spiked Plasma - Rep 2 114OOOO 434OOO 2.62 Spiked Plasma - Rep 3 112OOOO 437OOO 2.56 Spiked Plasma - Rep 4 126OOOO 434OOO 2.9 Spiked BSA - Rep 1 154OOO S62OOO O.274 O.275 O.O168 6.1 1143 Spiked BSA - Rep 2 142OOO S36OOO O.264 Spiked BSA - Rep 3 16OOOO S37OOO O.299 Spiked BSA - Rep 4 137000 S19 OOO O.263

Note: Average blank plasma area ration subtracted from spiked plasma area ratio before recovery was calculated, US 2015/03461 70 A1 Dec. 3, 2015 28

0214 FIGS.5A-5D and FIGS. 6A-6C provide exemplary methanol (0.5 mL) & a washing buffer (H2O: Acetonitrile: spectra for the thyroid hormones. Table 9 provide exemplary NHOH=85:15:1, 0.5 mL). Hydrolyzed urine sample (previ linear regression data for quantification of thyroid hormones. ous step, 0.5 mL) was added and passed through the column TABLE 9 Linearity - 33.1 pg/ml - 2ng/ml in BSA Analyte Peak IS Peak Area Calc. Conc. Sample Area (counts) (counts) Area. Ratio (pg/mL) R21 T3 31.3 pg/ml Rep 1 3,660 956,000 O.OO383 23 O.9994 T3 31.3 pg/ml Rep 2 3,910 240,000 O.OO314 11.9 T3 62.5 pg/ml Rep 1 7,500 420,000 O.OOS28 46.6 T3 62.5 pg/ml Rep 2 5,720 998,000 O.OOS/4 S4 T3125 pg/ml Rep 1 9,740 ,030,000 O.O09S 115 T3125 pg/ml Rep 2 9,610 916,000 O.O1 OS 131 T3 250 pg/ml Rep 1 18,800 ,000,000 O.O188 266 T3 250 pg/ml Rep 2 20,700 ,150,000 O.018 2S4 T3 500 pg/ml Rep 1 36,300 ,020,000 0.0357 S41 T3 500 pg/ml Rep 2 34,500 ,050,000 O.O328 494 T3 1 ng/ml Rep 1 64,400 ,010,000 O.0635 993 T3 1 ng/ml Rep 2 69,100 ,060,000 O.06SS 1030 T32 ng/ml Rep 1 103,000 843,000 O.122 1940 T32 ng/ml Rep 2 116,000 912,000 O.127 2040 rT331.3 pg/ml Rep 1 1,060 956,000 O.00111 24.1 rT331.3 pg/ml Rep 2 1,760 240,000 O.OO142 31.6 rT3 62.5 pg/ml Rep 1 3,400 420,000 O.OO239 55.2 rT3 62.5 pg/ml Rep 2 3,530 998,000 O.OO3S4 83.1 rT3125 pg/ml Rep 1 5,380 ,030,000 O.OOS25 124 rT3125 pg/ml Rep 2 4,890 916,000 O.OOS34 127 rT3 250 pg/ml Rep 1 11,400 ,000,000 O.0114 273 rT3 250 pg/ml Rep 2 10,300 ,150,000 O.OO902 216 rT3500 pg/ml Rep 1 22,900 ,020,000 O.O225 543 rT3500 pg/ml Rep 2 22,200 ,050,000 O.O211 509 rT3 1 ng/ml Rep 1 40,700 010,000 0.04.01 970 rT3 1 ng/ml Rep 2 41,700 ,060,000 O.O395 956 rT32 ng/ml Rep 1 76,700 843,000 O.091 2200 rT32 ng/m. Rep 2 68,500 912,000 0.0751 1820 T431.3 pg/ml Rep 1 1,550 956,000 O.OO162 37.7 O.9966 T431.3 pg/ml Rep 2 1,740 240,000 O.OO14 28.2 T4 62.5 pg/ml Rep 1 2,700 420,000 O.OO19 49.7 T4 62.5 pg/ml Rep 2 2,240 998,000 O.00224 64.1 T4 125 pg/ml Rep 1 4,390 ,030,000 O.OO428 152 T4 125 pg/ml Rep 2 3,670 916,000 O.004 140 T4250 pg/ml Rep 1 7,030 ,000,000 O.007 268 T4250 pg/ml Rep 2 7,660 ,150,000 O.OO668 2S4 T4500 pg/ml Rep 1 12,900 ,020,000 O.O127 512 T4500 pg/ml Rep 2 12,500 ,050,000 O.O119 477 T4 1 ng/ml Rep 1 23,800 ,010,000 O.O234 972 T4 1 ng/ml Rep 2 24,000 ,060,000 O.0228 943 T42 ng/ml Rep 1 43,000 843,000 O.OS1 21SO T42 ng/ml Rep 2 40,900 912,000 O.0448 1890 T3 62.5 pg/ml Rep 2 5,720 998,000 O.OOS/4 S4 Quadratic Regression (1ix weighing) of Standard Curve

Example 4 in 2 min under a nitrogen pressure (<15 psi). The column was washed sequentially with the washing buffer (H2O: Acetoni Derivatized THC trile:NHOH=85:15:1, 0.5 mL), methanol (0.5 mL), ethyl acetate, then dried under a nitrogen stream at 30 psi for 3 min. 0215. The presently recited methods were also performed The analyte is then eluted with the elution buffer hexane: on THC. The THC was derivatized during elution from the ethyl acetate:acetic acid (Glacial)=80:18:2, 100 uL), and SPE column as follows: dried under a nitrogen stream (40°C.). Quantification of 0218 Resuspend the dried extracted analyte with Fluore 11-nor-9-carboxy-delta-9-tetrahydrocannabinol in nylmethyloxycarbonyl chloride (FMOC-CI, 1.6 mg/mL in ACN, 50 uL), vortexed, then, potassium carbonate (aq., 100 Urine Method and Example Data mM, 50 ul) was added, vortexed. Reaction was allowed for 25 0216 Urine hydrolysis: Combine urine sample (2 mL) minutes, then suspended with a stopping buffer (NHCOH with KOH (aq., 10M, 100 uL) at 60°C. for 15min, add IS, and (aq. 50 mM):acetonitrile=1:4,10 pl. allow to cool to room temperature. Centrifuge at 2000 rpm for 0219. For LC-MS/MS analysis, 20 uL of the solution of 2 minutes. The clear Solution was taken to the next step. the derivatization reaction mixture was injected into a 0217 Solid phase extraction column narrow bore backed TARGAR) C183 um particle size 50x2.1 mm analytical col with sorbent (10BPC SAX, 2.5 mg) was equilibrated with umn. A binary HPLC gradient was applied to the analytical US 2015/03461 70 A1 Dec. 3, 2015 29 column to separate the thyroid hormones from other analytes TABLE 11 contained in the sample. Mobile phase A was 20.0 mM ammonium formate pH 3.0 and mobile phase B was Aceto Linearity nitrile with 0.1% formic acid. The HPLC gradient proceeded Analyte at a temperature of 40°C. with a flow rate of 500 ul/min over Peak Area Conc. Calculated - five minutes as follows: 0.01 min, 40% B;3.0 min, 90% B; 4.0 Sample Name (counts) (pg/mL) Conc. (pg/mL) R21 min, 90% B; 4.1 min, 40% B, 5.0 min, 40% B. SampleO15 ling 112OOOO 1OOO 1OOO 1.OOOO 0220 MS/MS was performed using an API 5000 triple Std 10Oul rxn 50-50 quadrupole mass spectrometer controlled by Analyst Soft SampleO16 100pg 147OOO 1OO 100 ware Version 1.52 (ABI-SCIEX, Toronto, Canada). Analyte Std 10Oul rxn 50-50 exiting the HPLC analytical column through the mobile SampleO17 10pg 186OO 10 10 phase flowed to the heated nebulizer interface of the MS/MS Std 10Oul rxn 50-50 analyzer. The solvent/analyte mixture was converted to vapor Quadratic Regression (1ix weighing) of Standard Curve in the heated tubing of the interface. Analytes in the nebulized solvent were ionized by heated Electrospray Ionization 0223 FIGS. 8A (standard) and 8B (the extracted sample) SOUC. show the chromatogram of the recovery of the analyte. This 0221) Ions passed to the first quadrupole (Q1), which data is also reflected in Table 12. selected ions with a mass to charge ratio of parent ions gen erated from one of the analytes. Ions entering quadrupole 2 TABLE 12 (Q2) collided with argon gas to generate ion fragments, which were passed to quadrupole 3 (Q3) for further selection. After Recovery of Analyte measurement of ions indicative of one of the analytes, Q1 was Peak Area Recovery adjusted so that ions with a mass to charge ratio of parention Sample Name (counts) (%) from a second analyte were selected. These ions were col lided with argon gas in Q2, and the ion fragments passed to SampleO08 1ng std 313 OOO 500ul 50-50ACN4ul CMOS 700tem Q3 for further selection. The following mass transitions were SampleO13 1ng spike 500ul 267OOO 85.3 used for detection and quantitation of the thyroid hormones 50-50ACN 4-ul CMOS 700tem during validation on positive polarity from the same sample injection. QC) HO

a O HOC O ={ C OH O OO HOC O Chemical Formula:CH' C14H11" Chemical Formula: C2H26O4" Exact Mass: 179.09 Exact Mass: 345.21 C O

Chemical Formula: C3H4NO" Exact Mass: 584.30

TABLE 10 0224. In view of the data presented herein, it is clear that the process simultaneously provides following benefits: short Ions Monitored sample handling time, automation compatible, chemical pro tection via derivatization over oxidation, which resulted in Precursor high recovery in extraction; improvement in HPLC behavior Compound Polarity mz Product miz due to high lipophilicity of the derivatives; and much THC-9-Carboxylic (+) Quantitative S84.2 3455 improved MS/MS sensitivity due to much high molecular acid (+) Confirmatory S84.2 1793 weight of the derivative, with LLOQ at 10 pg/mL, i.e. over 1000 fold improvement in quantification sensitivity. These benefits are unexpected in view of the high pH elution, as the 0222 Table 11 shows the quadratic linear regression for of stability of catecholamines declines as the pH rises, with 11-nor-9-carboxy-delta-9-tetrahydrocannabinol in Urine at destruction becoming extremely rapid in analkaline medium. 10 to 1,000 ng/reactions. FIGS. 7A-7C show the chromato 0225. It is also realized that the employment of high MS graphs of example linearity data at 1000 pg/ml, 100 pd/ml, sensitive derivatization allows one to screen, optimize the and 10 pg/ml. process of Solid phase extraction, including the choice of US 2015/03461 70 A1 Dec. 3, 2015 30 sorbent, loading buffer, washing solution, final elution buffer, term “about in the context of the mass of an ion or the as well as the process of each step. mass/charge ratio of an ion refers to +/-0.50 atomic mass 0226. In closing, it is to be understood that although unit. aspects of the present specification are highlighted by refer 0230. At the very least, and not as an attempt to limit the ring to specific embodiments, one skilled in the art will application of the doctrine of equivalents to the scope of the readily appreciate that these disclosed embodiments are only claims, each numerical indication should at least be construed illustrative of the principles of the subject matter disclosed in light of the number of reported significant digits and by herein. Therefore, it should be understood that the disclosed applying ordinary rounding techniques. Subject matter is in no way limited to a particular methodol 0231. Use of the terms “may' or "can' in reference to an ogy, protocol, and/or reagent, etc., described herein. As such, embodiment or aspect of an embodiment also carries with it various modifications or changes to or alternative configura the alternative meaning of “may not or “cannot.” As such, if tions of the disclosed subject matter can be made in accor the present specification discloses that an embodiment or an dance with the teachings herein without departing from the aspect of an embodiment may be or can be included as part of spirit of the present specification. Lastly, the terminology the inventive subject matter, then the negative limitation or used herein is for the purpose of describing particular exclusionary proviso is also explicitly meant, meaning that an embodiment or an aspect of an embodiment may not be or embodiments only, and is not intended to limit the scope of cannot be included as part of the inventive subject matter. In the present invention, which is defined solely by the claims. a similar manner, use of the term “optionally in reference to Accordingly, the present invention is not limited to that pre an embodiment or aspect of an embodiment means that Such cisely as shown and described. embodiment or aspect of the embodiment may be included as 0227 Certain embodiments of the present invention are part of the inventive subject matter or may not be included as described herein, including the best mode knownto the inven part of the inventive subject matter. Whether such a negative tors for carrying out the invention. Of course, variations on limitation or exclusionary proviso applies will be based on these described embodiments will become apparent to those whether the negative limitation or exclusionary proviso is of ordinary skill in the art upon reading the foregoing descrip recited in the claimed subject matter. 0232. Notwithstanding that the numerical ranges and val tion. The inventor expects skilled artisans to employ Such ues setting forth the broad scope of the invention are approxi variations as appropriate, and the inventors intend for the mations, the numerical ranges and Values set forth in the present invention to be practiced otherwise than specifically specific examples are reported as precisely as possible. Any described herein. Accordingly, this invention includes all numerical range or value, however, inherently contains cer modifications and equivalents of the Subject matter recited in tain errors necessarily resulting from the standard deviation the claims appended hereto as permitted by applicable law. found in their respective testing measurements. Recitation of Moreover, any combination of the above-described embodi numerical ranges of values herein is merely intended to serve ments in all possible variations thereof is encompassed by the as a shorthand method of referring individually to each sepa invention unless otherwise indicated herein or otherwise rate numerical value falling within the range. Unless other wise indicated herein, each individual value of a numerical clearly contradicted by context. range is incorporated into the present specification as if it 0228 Groupings of alternative embodiments, elements, or were individually recited herein. steps of the present invention are not to be construed as 0233. The terms “a” “an,” “the and similar referents used limitations. Each group member may be referred to and in the context of describing the present invention (especially claimed individually or in any combination with other group in the context of the following claims) are to be construed to members disclosed herein. It is anticipated that one or more cover both the singular and the plural, unless otherwise indi members of a group may be included in, or deleted from, a cated herein or clearly contradicted by context. All methods group for reasons of convenience and/or patentability. When described herein can be performed in any suitable order any Such inclusion or deletion occurs, the specification is unless otherwise indicated herein or otherwise clearly con tradicted by context. The use of any and all examples, or deemed to contain the group as modified thus fulfilling the exemplary language (e.g., “such as') provided herein is written description of all Markush groups used in the intended merely to better illuminate the present invention and appended claims. does not pose a limitation on the Scope of the invention 0229. Unless otherwise indicated, all numbers expressing otherwise claimed. No language in the present specification a characteristic, item, quantity, parameter, property, term, and should be construed as indicating any non-claimed element So forth used in the present specification and claims are to be essential to the practice of the invention. understood as being modified in all instances by the term 0234 Specific embodiments disclosed herein may be fur “about.” As used herein, the term “about” means that the ther limited in the claims using consisting of or consisting characteristic, item, quantity, parameter, property, or term so essentially of language. When used in the claims, whether as qualified encompasses a range of plus or minus ten percent filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in above and below the value of the stated characteristic, item, the claims. The transition term “consisting essentially of quantity, parameter, property, or term. Accordingly, unless limits the scope of a claim to the specified materials or steps indicated to the contrary, the numerical parameters set forthin and those that do not materially affect the basic and novel the specification and attached claims are approximations that characteristic(s). Embodiments of the present invention so may vary. For instance, as mass spectrometry instruments can claimed are inherently or expressly described and enabled vary slightly in determining the mass of a given analyte, the herein. US 2015/03461 70 A1 Dec. 3, 2015

0235 All patents, patent publications, and other publica wherein R', R. R. and R are each independently, H, tions referenced and identified in the present specification are fluorine, chlorine, bromine, iodine, a cyano group, an individually and expressly incorporated herein by reference acetylene group, a propylene group, a Vinyl group, a in their entirety for the purpose of describing and disclosing, linear Cs alkyl, a branched Cls alkyl, a cyclic Cs for example, the compositions and methodologies described alkyl, a Substituted vinyl group, a linear Cs alkoxy, a in Such publications that might be used in connection with the branched Cls alkoxy or cyclic Cls alkoxy. present invention. These publications are provided solely for 8. The method according to claim 7, wherein the acyl their disclosure prior to the filing date of the present applica chloride is one of the following compounds: tion. Nothing in this regard should be construed as an admis sion that the inventors are not entitled to antedate such dis closure by virtue of prior invention or for any other reason. All R1 O statements as to the date or representation as to the contents of these documents is based on the information available to the N - - O - C applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

1. A method for determining the presence one or more O analytes in a test sample, the method comprising: RI O a) Solid phase extracting of the one or more analyte from the test sample, R2 ls b) eluting the one or more analytes from the Solid phase O C extraction with an elution Solution having a basic pH, or an acidic pH, or a neutral pH, R7 c) in situ derivatizing of the one or more analytes with a derivatizing agent, and d) detecting the one or more derivatized analytes using liquid chromatography and/or mass spectrometry. wherein the fused ring system is an all carbocyclic or a 2. The method according to claim 1, wherein the one or heterocyclic aromatic system, optionally a naphthalene, more analytes is a compound having a primary amine, a a quinolone, an isoquinoline, a quinzoline, a benzofuran, secondary amine, or a phenolic hydroxyl group. an indole, or a benzimidazole, with a 5-7 membered 3. The method according to claim 2, wherein the one or Substituted or unsubstituted fused ring, or a nonaromatic more analytes is a monoamine neurotransmitter including carbocyclic, or heterocyclic fused ring system such as catecholamine or one of its derivatives or metabolites, a sex 3,4-dihydro-1H-indene, or a 3.4.dihydro-2H-chromene, hormone or one of its derivatives or metabolites, a cannab with a 4-8 membered fused ring, wherein the fused ring inoid or one of its derivatives or metabolites, a thyroid hor may be substituted when appropriate by one or more of mone or one of its derivatives or metabolites, an opiate, opioid a halogenatoms, optionally fluorine, chlorine, bromine, or one of its derivatives or metabolites or an arylcyclohexy or iodine, a cyano group, an acetylene group, a propy lamine or one of its derivatives or metabolites, an amphet amine or one of its derivatives or metabolites. lene group, a vinyl group, a linear Cs alkyl, a branched 4. The method according to claim 1, wherein the sample is Cs alkyl, a cyclic Cls alkyl, a substituted vinyl group, a biological sample, a Soil sample, or a sample of food stuff. a linear Cs alkoxy, a branched Cls alkoxy or cyclic 5. The method according to claim 1, wherein the derivatiz Cs alkoxy. ing reagent is an acyl halide. 9. The method according to claim 7, wherein the acyl 6. The method according to claim 5, wherein the acyl chloride is one of the following compounds: halide is an acyl chloride. 7. The method according to claim 6, wherein the acyl chloride is a compound of Formula II: (III) (II) ---,

wherein R' and R are each independently, H, fluorine, chlorine, bromine, iodine, a cyano group, an acetylene group, a propylene group, a Vinyl group, a linear Cls US 2015/03461 70 A1 Dec. 3, 2015 32

alkyl, a branched Cls alkyl, a cyclic Cls alkyl, a Sub- 12. The method according to claim 11, wherein the acyl stituted vinyl group, a linear Cls alkoxy, a branched chloride is one of the following compounds: Cs alkoxy or cyclic Cls alkoxy. R1 O RI O 10. The method according to claim 9, wherein the acyl R2 R2 chloride is one of the following compounds: C or C.

O

wherein the fused ring system may be an all carbocyclic or () 1s, a heterocyclic aromatic system, having a 5-7 membered fused ring, or a nonaromatic carbocyclic, or heterocyclic fused ring system such as 3,4-dihydro-1H-indene, or a 3,4-dihydro-2H-chromene, or having a 4-8 membered fused ring, and wherein the fused ring may be substi tuted when appropriate with one or more of a halogen atom, a cyano group, an acetylene group, a propylene O group, a Vinyl group, a linear Cls alkyl, a branched C1s ls alkyl, a cyclic Cls alkyl, a Substituted vinyl group, a linear Cs alkoxy, a branched Cls alkoxy or cyclic Cs C alkoxy. 8. chloride13. The is themethod following according compound: to claim 11, wherein the acyl O o 1s, B r

O ls 14. The method according to claim 6, wherein the acyl () O C. chloride is a compound of Formula VIII: (VIII)

R2 C Br R3 R5 11. The method according to claim 6, wherein the acyl R4 chloride is a compound of Formula VII: wherein R. R. R. R. and Rare each independently H, (VII) a halogen atom, optionally, fluorine, or a chlorine, or a R1 O bromine, or an iodine, a cyano group, an acetylene R2 ls group, a propylene group, a Vinyl group, a linear Cls O C alkyl, a branched Cls alkyl, a cyclic Cls alkyl, a Sub stituted vinyl group, a linear Cs alkoxy, a branched 3 5 Cs alkoxy or cyclic Cls alkoxy. R R 15. The method according to claim 14, wherein the acvl R4 chloride is one of the following compounds: R1 O RI O wherein R. R. R. R', and Rare each independently H, R2 R2 a halogen atom such as a fluorine, or a chlorine, or a C or C. bromine, or an iodine, a cyano group, an acetylene group, a propylene group, a Vinyl group, a linear Cls R3 R7 alkyl, a branched Cls alkyl, a cyclic Cls alkyl, a sub stituted vinyl group, a linear Cs alkoxy, a branched Cs alkoxy or cyclic Cls alkoxy. US 2015/03461 70 A1 Dec. 3, 2015 33

wherein the fused ring system may be an all carbocyclic or bromine, or iodine, a cyano group, an acetylene group, a a heterocyclic aromatic system, having a 5-7 membered propylene group, a Vinyl group, a linear Cls alkyl, a fused ring, or a nonaromatic carbocyclic, or heterocyclic branched Cls alkyl, a cyclic Cls alkyl, a Substituted fused ring system such as 3,4-dihydro-1H-indene, or a vinyl group, a linear Cs alkoxy, a branched Cisalkoxy 3,4-dihydro-2H-chromene, or having a 4-8 membered or cyclic Cisalkoxy. fused ring, and wherein the fused ring may be substi- 18. The method according to claim 17, wherein the acyl tuted when appropriate with one or more of a halogen chloride is one of the following compounds: atom, a cyano group, an acetylene group, a propylene group, a Vinyl group, a linear Cs alkyl, a branched Cs alkyl, a cyclic Cls alkyl, a Substituted vinyl group, a o O RI O O linear Cs alkoxy, a branched Cls alkoxy or cyclic Cs R2 N^ R2 N^ alkoxy. NC NC 16. The method according to claim 14, wherein the acyl chloride is the following compound: R3 R7

O O wherein the fused ring system may be an all carbocyclic or C. a heterocyclic aromatic system, optionally a naphtha lene, a quinolone, an isoquinoline, a quinzoline, a ben Zofuran, an indole, or a benzimidazole, having a 5-7 membered fused ring, or a nonaromatic carbocyclic, or heterocyclic fused ring system optionally 3,4-dihydro 17. The method according to claim 6, wherein the acyl 1H-indene, 3,4-dihydro-2H-chromene, having a 4-8 chloride is a compound of Formula IX: membered fused ring, and wherein the fused ring may be Substituted when appropriate by one or more of a halo gen atoms, optionally fluorine, chlorine, bromine, R O O (IX) iodine, a cyano group, an acetylene group, a propylene V/ group, a vinyl group, a linear C1s alkyl, a branched C1-8 R2 Sn alkyl, a cyclic Cls alkyl, a substituted vinyl group, a C linear Cs alkoxy, a branched Cls alkoxy or cyclic Cs alkoxy. R3 R5 19. The method according to claim 1, wherein the mass spectrometry comprises a tandem mass spectrometry tech R4 nique or a LC-MS/MS technique. 20. The method according to claim 1, wherein the detection wherein R. R. R. R', and Rare each independently, a of the one or more analytes is qualitative or quantitative. hydrogen, a halogen atom, optionally fluorine, chlorine, k . . . .