Standard-Free Bioanalytical Approach for Absolute Quantitation of

Jul 11, 2017 - Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stabl...
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A Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stable Isotopologues and Its Application Yong Gong, Jie Chen, Yifan Shi, Heng-Keang Lim, Naidong Weng, and Rhys Salter Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01830 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Analytical Chemistry

A Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stable Isotopologues and Its Application Yong Gong,* Jie Chen, Yifan Shi, Heng-Keang Lim, Naidong Weng, and Rhys Salter Department of Pharmacokinetics Dynamics & Metabolism, Janssen Research & Development, Johnson & Johnson, Welsh & McKean Roads, Spring House, Pennsylvania 19477, United States ABSTRACT: The following work describes a combined enzymatic and bioanalytical method that permits absolute quantitation of metabolites in biological samples without the requirement for reference metabolite standards. This technique was exemplified using a radio (14C) isotopologue and a stable (13C6) isotopologue of acetaminophen as substrates for in vitro biosynthesis of the corresponding radio and stable isotope labeled metabolites, namely, 14C- and 13C6- glucuronides and sulfates. By supplanting the use of authentic metabolite standards, traditionally used to calibrate 13C6-metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS), 13C6-metabolites were radiocalibrated by their 14C-isotopologues via liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS). The radiocalibrated 13C6-isotopologues were in turn used to quantitate acetaminophen and its corresponding metabolites in rat plasma samples by LC-MS/MS. Variation between this and a conventional LCMS/MS method using authentic standards for calibration was within ± 17%, permitting its use in preclinical and clinical applications. Since authentic metabolite standards are not required under the concept of radio and stable isotopologues using adapted LCRAD/MS protocols, significantly fewer resources are required to support accurate metabolite quantitation which in turn enables efficient analysis of simple and complex metabolite profiles.

Quantitation of circulating drug and its metabolites is an essential part of drug development. Determination of exposure levels of the drug and its putative metabolites in preclinical species provides essential pharmacokinetic and metabolism information for the selection of species in the toxicology assessment and assures the appropriate safety margins are available for parent and metabolites before progression into clinical studies.1 Metabolite related safety concerns continue to be the subject of intense focus with the publication of several seminal articles over the past decade such as the guidance for metabolites in safety testing (MIST) from the US Food and Drug Administration (FDA),2 as well as the guidance from the International Conference on Harmonisation (ICH).3 The liquid chromatography-tandem mass spectrometry (LCMS/MS) quantitation4,5 of metabolites, which uses both authentic metabolites as reference standards and stable isotopologues (STILs), i.e. stable isotope labeled metabolites, as internal standards (IS), is a conventional bioanalytical method with high sensitivity, accuracy, precision, and throughput. For example, metabolites of acetaminophen6 from different clinical studies have been quantified by LC-MS/MS in the presence of metabolite standards with deuterated isotopologues as IS.7-9 However, during drug discovery and development, such a quantitation approach requires significant investment of time and resources in the synthesis, purification and characterization of different metabolites and STILs. Biogenerated STILs of metabolites offer comparable quantitative results as chemically synthesized STILs, and furthermore, save significant synthesis resources leading to timely availability of IS.10-11 However, authentic metabolites standards are still required for the construction of calibration curves.

Hence, in the absence of authentic standards, a number of fast and semi quantitative LC-MS based methods have been reported that support fit-for-purpose tiered bioanalytical strategies.12-18 The reported mixed matrix approach for animal-tohuman exposure comparison covered MIST without authentic standards.19-20 However, absolute concentrations cannot be obtained by this method and thus determination of metabolite exposure levels by other approaches is still needed. Biosynthesized and preparative HPLC purified metabolites can be quantified by NMR spectroscopy for early quantitative estimation of drug metabolites in human plasma. These quantified metabolite isolates can be used in pharmacologic and drug metabolism studies.21-22 Radio isotopologues (RADILs), e.g. radio isotope labeled drugs, are often used in the metabolic profiling of the drugs. 2325 For in vivo and clinical studies carried out with the cold/unlabeled drugs, RADILs of metabolites from in vitro/in vivo biotransformation of RADILs of drugs have been used in the estimation of drug metabolite concentrations by liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS).15-18 However, the presence of unlabeled analytes in RADILs and the overlapping of isotopic distribution between RADILs and the analytes limit the dynamic range and accuracy of quantitation. In general, radio and stable isotopologues are normally generated and utilized separately (not just physically but in different departments within the traditional pharma model) in the support of quantitative analysis of drug metabolites by LCRAD/MS and LC-MS/MS, respectively. It would be advantageous if these two isotopologues could be used jointly in the quantitation of metabolites without the need for synthetic me1

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tabolite reference standards. To do so, the STILs should possess sufficient number of stable labels (e.g. M+6) to avoid interference with mass signals from the corresponding RADILs (e.g. M+2) and unlabeled analytes (M0). Radio labels (e.g. 14C) in RADILs can enable the radiocalibration process, in which RADILs calibrate STILs utilizing radioactivity counts to elicit the quantitation from mass spectrometry (MS) responses. Concentrations of RADILs can thus be determined using liquid scintillation counting (LSC) and LC-RAD/MS. Concentrations of STILs can be subsequently determined from radiocalibration of MS responses of STILs. The radiocalibrated STILs can then be used to quantify metabolites in biological samples using LC-MS/MS without the need of authentic metabolite standards for the construction of calibration curves as in traditional bioanalytical methods. Only the STILs and RADILs of parent drugs require chemical synthesis, which are often available during preclinical drug development. The related STILs and RADILs of metabolites can be generated from the labeled parents using in vitro biotransformation conditions developed during preclinical drug profiling and used directly in the metabolite quantitation via a LC-RAD/MS protocol. This would effectively eliminate synthetic campaigns for metabolite standards. To validate this new approach, acetaminophen (APAP) and its sulfate (Sulf) and glucuronide (Gluc) metabolites were selected for quantitation based on their availability, stability, and known in vitro metabolic profiles. Moreover, Sulf and Gluc are the major systemic metabolites in human. Two isotopologues of acetaminophen, 14C-acetaminophen (14C-APAP) and 13 C6-acetaminophen (13C6-APAP), were synthesized separately followed by incubation to form 14C- and 13C6- Sulf and Gluc (Scheme 1). The crude mixture of 13C6-isotopologues from the incubation were radiocalibrated by 14C-isotopologues from the incubation by LC-RAD/MS, and used in the quantitation of APAP and its two metabolites in rat plasma by LC-MS/MS. The results were in turn compared to a conventional LCMS/MS method using authentic metabolite standards. Scheme 1. Quantitation protocol of acetaminophen (APAP) and its metabolites (Sulf and Gluc)

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Materials. Acetaminophen, acetaminophen sulfate potassium salt and acetaminophen glucuronide sodium salt were purchased from Sigma-Aldrich (Milwaukee, WI, USA). 14CAcetaminophen (radiochemical purity >99%, specific activity 59 millicurie (mCi)/mmol, specific concentration 0.5 mCi/mL in ethanol) was supplied by ViTrax Co. (Placentia, CA, USA). 13 C6-Acetaminophen (isotopic purity 99 atom% 13C) was prepared from 13C6-4-aminophenol and acetic anhydride according to synthetic reports for 14C-acetaminophen.26,27 Uridine 5’diphospho-N-acetylglucosamine sodium salt (UDPAG), uridine 5’-diphosphoglucuronic acid ammonium salt (UDPGA), adenosine 3’-phosphate 5’-phosphosulfate lithium salt hydrate (PAPS), 1,4-dithiothreitol (DTT), alamethicin, formic acid, ammonium hydroxide and ammonium formate (Sigma Ultra grade) were from Sigma-Aldrich (St. Louis, MO, USA). Male monkey S9 (lot AAL) was from Celsis/In Vitro Technologies (Baltimore, MD, USA). HPLC grade acetonitrile, methanol and isopropyl alcohol were from EMD Chemicals, Inc (Gibbstown, NJ, USA). HPLC grade water was obtained from Burdick and Jackson (Muskegon, MI, USA). Instrumentation. The LC-RAD/MS system consisted of a Thermo Scientific Accela LC (San Jose, CA, USA), coupled to a Thermo Scientific LTQ and a v. ARC radioactivity detector from AIM Research Company (Hockessin, DE, USA). Data acquisition and reduction was carried out using XcaliburTM 2.0 from Thermo Scientific (San Jose, CA, USA). The LC-MS/MS system consisted of a Shimadzu LC-20AD liquid chromatograph (Kyoto, Japan), coupled with a Shimadzu SIL20AC autosampler and an AB Sciex API 5000 (Foster City, CA, USA) triple quadrupole mass spectrometer with a TurboIonSpray interface in negative ionization mode. The system was controlled by Analyst 1.6.2. In vivo Samples. The animal experiments were carried out in accordance with the guide for the care and use of laboratory animals. APAP was formulated in demineralized water containing 0.5% w/v methocel as an aqueous suspension at 100 mg/mL concentration. Three male Sprague-Dawley rats were dosed orally with APAP at 500 mg/kg. The blood samples (300 µL) at pre-dose (0 hour), 1, 2, 4, 6 and 24 hours were collected via the saphenous vein into K3EDTA coated Sarstedt Microvette® tubes, and then placed on wet ice. Within 1 hour of collection, the samples were centrifuged (2,000 × g, 4 minutes, 4 °C). The plasma samples (150 µL) were then transferred to a clean 96-well plate, frozen, and stored in a −70 °C freezer. In vitro incubation. The 14C-metabolites and 13C6metabolites were generated from parallel relay incubation of the 14C-APAP and 13C6-APAP in monkey S9 by adaptation of previously reported procedure28 to increase the production of the metabolites. The incubation was carried out at 37 °C with PAPS (20 µM) and UDPGA/UDPAG (5/1 mM) as cosubstrates in 0.1 M phosphate buffer (pH 7.4), containing dithiothreitol (1 mM), alamethicin (25 µg/ml), monkey S9 (1 mg/mL) and 14C- or 13C6- APAP (50 µM). The total volume was 5 mL. After incubation for 2 hours, the mixture was centrifuged (200,000 × g, 15 minutes, 4 °C) to remove the S9 protein pellets. Fresh S9 and cofactors were added to the supernatant for each relay incubation. After three consecutive relay incubations, the incubation mixtures were quenched with 3 volumes of ice-cold acetonitrile containing 0.02% formic acid, vortexed and sonicated (3 minutes). Precipitated proteins were pelleted by centrifugation (3,000 × g, 10 minutes, 4 °C). The supernatants were evaporated down to approximately 2

EXPERIMENTAL SECTION 2

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mL under a gentle stream of nitrogen. The concentrated biosynthesized samples were used as stock solutions. The metabolic profiles of concentrated biosynthesized samples were analyzed by LC-RAD/MS. The 14C- and 13C6- isotopologues of APAP, Sulf and Gluc were three major components identified in the corresponding biosynthesized samples. Determination of Radioactivity. The total radioactivity of the 14C-stock solution was determined by LSC. A 20 µL aliquot of 14C-stock solution was diluted 10-fold by addition of 180 µL acetonitrile/water 9:1. Three aliquots (3 × 50 µL) of the diluted sample were added to three 10 mL scintillation vials containing 50 volumes of liquid scintillation cocktail, and counted in a Tri-Carb 3110TR liquid scintillation analyzer (Perkin Elmer Life and Analytical Science; Waltham, MA, USA) in triplicates. The average radioactivity of the 14C-stock solution was obtained in the unit of disintegrations per minute (dpm). The percentage radioactivity of each 14C-isotopologue in the stock solution was determined by LC-RAD/MS.

RESULTS AND DISCUSSION Design of Isotopologues. Non-exchangeable radio (14C) and stable (13C) isotope labels at metabolically stable positions are preferred to avoid potential exchange and loss of isotope labels during the biotransformation, as well as potential deuterium isotope effects.29 One 14C label with high specific activity should reach double digit ng/mL level radio detection limit.30 RADILs and STILs of metabolites can be quickly accessed without extensive purification via in vitro biotransformation of corresponding isotopologues of parent drug. This is only feasible if there is significant formation of the metabolite by the biotransformation pathway. For 14C-APAP, the specific activity from 14C label should be a constant and carried over to the 14C-Sulf and 14C-Gluc metabolites. The amount of 14C-APAP and 14C-metabolites can be quantitated by LC-RAD/MS and subsequently used for radiocalibration of MS responses of corresponding STILs. The STILs should be free from mass interference by isotopic peaks not only from the analytes, but also from the RADILs. For example, six 13C labels in 13C6-APAP, 13C6-Sulf and 13C6-Gluc are sufficient and ensure the mass separation between 13C6isotopologues (M+6) and 14C-isotopologues (M+2), as well as 13 C6-isotopologues (M+6) and analytes (M0), and therefore, avoiding cross-talk in each mass channels during LCRAD/MS and LC-MS/MS analysis. Biogeneration of RADILs and STILs of Metabolites. The selection of in vitro system for generation of isotopologues of metabolites can be based on the known in vitro profiles of parent drugs cross species to ensure generation of adequate amount of RADILs and STILs of metabolites. The careful selection of the labels and labeling positions will ensure both radio and stable labels remain intact in the target metabolites. In the presence of PAPS and UDPGA, satisfactory turnovers of 14C- and 13C6- APAP to 14C- and 13C6- Sulf and Gluc were achieved separately from relay incubation28 (3 × 2 hours) with monkey S9. A radio chromatogram of incubation solution containing 14C-APAP, 14C-Sulf and 14C-Gluc is shown in Figure 1. The radio response was measured in counts per minute (CPM). A baseline separation of metabolites and high recovery of radioactivity (>95%) were critical for the accuracy of quantitation. A similar distribution of 13C6-APAP, 13C6-Sulf and 13C6-Gluc in the STIL stock solution was also obtained. The stock solutions containing 14C-isotopologues and 13C6isotopologues were used without further purification.

Standards and Solutions. A 200 µL aliquot of 14C- and C6-stock solutions were mixed and diluted with equal volume of acetonitrile containing 0.02% formic acid. The mixture was filtered through a 0.45 µm nylon filter by centrifugation (10,000 × g, 2 minutes, at room temperature). The filtrate was analyzed in triplicate by LC-RAD/MS for the determination of concentration of 13C6-APAP, 13C6-Sulf and 13C6-Gluc in biosynthesized 13C6-stock solution. The radiocalibrated 13C6-stock solution was diluted six times with methanol to make a 13C6-working solution without any further purification. This 13C6-working solution with known final concentrations for 13C6-APAP, 13C6-Sulf and 13C6-Gluc was spiked into rat plasma samples to quantify APAP and its metabolites based on the corresponding peak areas of unlabeled analytes and 13C6-isotopologues. A set of calibration standards and quality control (QC) samples using authentic analytes were prepared to validate the results obtained by the LC-RAD/MS method. Stock solutions of APAP, Sulf and Gluc were prepared at a concentration of 1.00 mg/mL in 50:50 methanol/water (v/v). The STIL used in the LC-RAD/MS method served as the IS in the conventional LC-MS/MS method. Eight calibration standards covering the range from 10.0 to 5000 ng/mL were prepared by serial dilution in rat plasma. Four levels of QCs at the concentrations of 30.0, 150, 2500, and 4000 ng/mL were used for the evaluation of the ruggedness of the extraction and chromatographic methods for APAP, Sulf and Gluc. All prepared solutions, unknown samples, calibration standards, and QC samples were stored at −70 °C. Preparation of Analytical Samples. Rat plasma samples were processed similarly as described in the literature.9 Prior to protein precipitation, samples at 1, 2, 4 and 6 h time points were diluted 50 times with blank rat plasma, and samples at 24 h time point were diluted 5 times with blank rat plasma. An aliquot of 25 µL diluted plasma was pipetted into a well of a 96-deep-well plate, and 25 µL of STIL working solution was added to each sample followed by 150 µL of methanol. The mixture was vortexed, and then centrifuged (2,500 × g, 5 minutes, 4 °C). Aliquots of 150 µL of the supernatant were transferred, evaporated to dryness at 40 °C, and then reconstituted with 150 µL of 0.01% formic acid in water. The samples were analyzed concurrently with authentic standards and STILs using LC-MS/MS method. 13

9000

[14 C]APAP

8000 7000 Response (CPM)

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6000 5000 4000 3000 2000

[14 C]Sulf [14 C]Gluc

1000 0 0

4

8

12

16 Time (min)

20

24

28

32

Figure 1. A radio chromatogram of 14C-stock solution

In this study, 14C-APAP and 13C6-APAP at sub-µmol level were used separately in the incubation. Only a fraction (µCi level) of resulting 14C-isotopologues in 14C-stock solution was 3

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final concentrations of 13C6-APAP, 13C6-Sulf and 13C6-Gluc listed in Table 2. This 13C6-working solution was used for the quantitation of APAP and its metabolites using LC-MS/MS method without the need of calibration curves. Table 2. Radiocalibration of STILs

needed for radio quantitation and radiocalibration. The amount of 13C6-isotopologues in 13C6-stock solution was sufficient for subsequent calibration and quantitation. Radio Quantitation of RADILs in the Stock Solution. The concentration of each RADIL (CRADIL) in the stock solution can be determined from the total radioactivity (RAtotal) of the stock solution, the percentage radioactivity (RA%) of each RADIL in the stock solution, and specific activity (SA) of each RADIL using eq 1.  =

 × % 









14

SA (dpm/ng)

C-APAP

907

14

C-Sulf

595

C-Gluc

422

14

RAtotal (dpm/µL) 12720 (± 110)

CRADIL (µg/mL)

69.4 ± 0.8

9.73

12.9 ± 0.2

2.76

17.7 ± 0.5

5.34

 

× 

1.168 ± 0.040

1.89

C6-Sulf

1.463 ± 0.090

0.673

C6-Gluc

1.226 ± 0.022

1.09

C6-APAP

13 a

Working solution after 6-fold dilution of radiocalibrated stock solution.

Conventional LC-MS/MS quantitation. A set of calibration standards and QC samples of APAP, Sulf and Gluc were analyzed in parallel with the unknown samples. Unknown samples were quantified using the calibration curve where the STILs from incubation served as the IS. The precision and accuracy of the LC-MS/MS method for APAP, Sulf and Gluc were assessed in a qualification batch consisting of four different concentrations (30.0, 150, 2500, 4000 ng/mL) of QC samples. All the accuracy and coefficient of variation (CV) were within ± 15%. The 13C6-working solution was added to the reference standards, QCs and blank as IS in the qualification batch. Thus, the peak area of each STIL was used in the calculation of area ratio of every reference standard and QC sample for qualification. The biogenerated 13C6-isotopologues provided acceptable performance as IS based on the precision and accuracy obtained across the calibration range. Although 13 C6-solution contained impurities from monkey S9 and byproducts from incubation, no interference to M0 of parent or metabolites was introduced by the biogenerated STILs in the chromatogram of blank spiked with 13C6-solution. Quantitation by LC-RAD/MS method. The concentrations of APAP, Gluc and Sulf (Canalyte) in rat plasma samples were calculated individually by eq 3, based on the radiocalibrated concentration of the corresponding STIL (CSTIL) from eq 2, and the MS peak area ratio between analyte and STIL (Aanalyte/ASTIL). Since plasma samples were diluted with a dilution factor (fdilu) of 50 for 1 h, 2 h, 4h, 6 h samples and 5 for 24 h, fdilu was also included in the eq 3.

Radiolysis, or secondary radiolytic self-decomposition, occurs commonly for all radio labeled compounds. While empirical, the rate of degradation generally increases with high concentrations, high specific activity, and the presence of impurities. It may also be affected by storage and working conditions. Hence, it is recommended that once biogenerated and unpurified RADIL in stock solution is radio quantified, the solution is used immediately thereafter. Re-quantitation of RADIL prior to use will minimize the impact on accuracy by the radiolytic effects. Radiocalibration of STILs in the Stock Solution. Each STIL in stock solution can be radiocalibrated with the corresponding RADIL by LC-RAD/MS. The concentration of each STIL (CSTIL) in the mixture can be calculated from the corresponding MS peak area ratio between each STIL and RADIL pair (ASTIL/ARADIL) and the radio quantified corresponding CRADIL from eq 1. Thus, the concentrations of 13C6-APAP, 13C6Sulf and 13C6-Gluc in the stock solution were calculated using eq 2.  =

CSTIL (µg/mL)a

13

(1)

RA%

ASTIL/ARADIL

STIL 13

SA is defined as the activity (radioactivity) per quantity of a particular radionuclide. A commonly used unit is the millicurie per millimole (mCi/mmol). Carbon-14 (14C) has a constant SA of 62.5 mCi/mmol, which is the maximum radioactivity value for a single 14C label. Actual SA of 59 mCi/mmol was determined for the 14C-APAP used in this study. The lower SA of 59 mCi/mmol reflected the presence of 5.6% unlabeled component in the RADIL. To avoid interference from unlabeled analyte in the RADIL, a SA of 62.5 mCi/mmol should be used in the calculation. The 14C-metabolites generated from S9 incubation with 14C-APAP should have the same SA as the unchanged 14C-APAP. For easy calculation, a SA value in mCi/mmol unit was converted to corresponding dpm/ng unit (1 mCi = 2.22 × 109 dpm), based on the molecular weight (MW) of 14C-APAP (MW 153), 14C-Sulf (MW 233) and 14CGluc (MW 329). Therefore, the concentrations of 14C-APAP, 14 C-Sulf and 14C-Gluc in the stock solution were calculated. The values are summarized in Table 1. This radio quantified 14 C-stock solution was used for the calibration of 13C6-stock solution. Table 1. Radio quantitation of RADILs RADIL

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 !"# =

 $ % & 

×  × '() *

(3)

The mean plasma concentration of three rats vs time plots for APAP, Sulf and Gluc are shown in Figure 2. The data set generated from LC-RAD/MS protocol using RADILs (14Cisotopologues) for the calibration of STILs (13C6isotopologues) matched closely with that from conventional LC-MS/MS method using authentic reference standards for calibration curves. With Canalyte from LC-MS/MS as a comparison standard, the percentage differences between the two methods were calculated as 16.6 ± 0.4 % for APAP, 13.5 ± 0.6 % for Sulf and -2.2 ± 0.9 % for Gluc. Since the same set of Aanalyte/ASTIL was used by both quantitation methods, the Canalyte variations between the two methods for each analyte indicated the degree of agreement between one point calibration by LCRAD/MS and standard curve calibration by LC-MS/MS method. Other factors, including the precision of radioactivity determination, purity of authentic standards, and stability of isotopologues, may potentially contribute to the observed variations between the two methods.

(2)

13

The radiocalibrated C6-stock solution was further diluted by six-fold with methanol to a 13C6-working solution with the 4

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1000

1000

1000 (b) Sulf

100

10

1

(c) Gluc

LC-RAD/MS LC-MS/MS

Concentration (µg/mL)

LC-RAD/MS LC-MS/MS

Concentration (µg/mL)

(a) APAP

Concentration (µg/mL)

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100

10

100

5

10 15 Time (hours)

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10

1

1 0

LC-RAD/MS LC-MS/MS

0

5

10 15 Time (hours)

20

25

0

5

10 15 Time (hours)

20

25

Figure 2. Plasma concentration vs time curves for (a) APAP, (b) Sulf and (c) Gluc. Error bars represent the standard deviation from three rats.

There are potential limitations and requirements of this approach. Labeling positions on the drugs and metabolites must be metabolically stable. Both radio and stable labels must remain in target RADILs and STILs of metabolites following their production through biotransformation. Accessibility to some target RADILs and STILs of drugs may be challenging. Unique and/or trace in vivo metabolites may be difficult to produce or inaccessible from in vitro conditions. Potential radiolysis of RADILs requires stability check or re-quantitation prior to use. Pharmacokinetics (PK). PK parameters were determined based on the data set from LC-RAD/MS and LC-MS/MS methods. The differences in area under the curve (AUC) between the two methods were 16.6% for APAP, 13.6% for Sulf and -2.3% for Gluc, which matched differences in peak plasma concentration (Cmax). There were no observed differences in time to reach Cmax (Tmax).

Corresponding Author * Phone: 215-628-6436. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We are grateful to David C. Evans and Wenying Jian for reviewing the manuscript, to William J. Kintigh for providing the rat plasma samples.

REFERENCES (1) (2) (3)

CONCLUSIONS

(4)

The concept of using a RADIL in the calibration of a STIL, and subsequent measurement of metabolite concentration without metabolite reference standard has been demonstrated in the example of LC-RAD/MS based quantitation of acetaminophen and its sulfate and glucuronide metabolites in rat plasma samples. In vitro incubation of a RADIL and a STIL of acetaminophen offered immediate access to the target RADIL and STIL of metabolites. The quantitation results were cross-validated using a conventional LC-MS/MS approach and metabolite reference standards for the construction of calibration curves. Variations of calculated concentrations and related PK data between the two methods were within ± 17% of each other. The generation and application of the reciprocal RADIL and STIL to the LC-RAD/MS method described herein is anticipated to meet bioanalytical needs by reducing resources needed and cycle time while preserving quantitation accuracy required for both preclinical and clinical studies.

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(6) (7) (8) (9) (10)

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website. LC-RAD/MS conditions, LC−MS/MS conditions, and analytical data (PDF)

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AUTHOR INFORMATION

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