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User Benefits Application News Sample Preparation Reaction solutions obtained after metabolic reactions by cytochrome P450 (CYP), with coumarin, diclofenac, and nifedipine added as substrates, were used as analytical samples. The sample preparation process is shown in Fig. 2. Introduction In pharmacokinetic studies, drug concentrations in biological samples are analyzed to clarify the absorption, distribution, metabolism, and excretion of drugs in vivo. Because rapid results are required and a large number of samples must be handled, faster analytical methods are increasingly important. This report describes an example of high-throughput analysis of six drug metabolites using a system consisting of a Nexera X4 ultra high-performance liquid chromatograph and a LCMS- 8060RX triple quadrupole mass spectrometer. The high-speed analysis time of 0.80 min contributes to higher throughput. Nexera X4’s excellent high-speed gradient performance in combination with the LCMS -8060RX CoreSpray technology enables consistent and reliable trace quantitative analysis. High-Throughput Analysis of Drug Metabolites Using a Triple Quadrupole LC-MS/MS System Miho Kawashima High Performance Liquid Chromatograph Mass Spectrometer Prepare 50 µL of microsome mixture - 0.2 mg/mL Human liver microsome - Substrates (coumarin and diclofenac 2 µmol/L, nifedipine 1 µmol/L) - 100 mM Potassium phosphate buffer (pH 7.4) Preincubate for 5 min at 37 °C Incubate for 10 min at 37 °C Add 50 µL of NADPH regeneration system - 6.6 mM Magnesium chloride - 6.6 mM Glucose-6-phosphate - 2.6 mM NADP+ - 0.8 U/ml Glucose-6-phosphate dehydrogenase - 100 mM Potassium phosphate buffer (pH 7.4) Add 300 µL cold Acetonitrile (Stop the reaction) Centrifuge with 10,000 g for 10 min Dispense 150 µL of supernatant for measurement Analysis Conditions The LC-MS/MS analysis conditions are indicated in Table 1. The target compounds and MRM conditions are indicated in Table 2. HPLC Conditions System: Nexera X4 Column: Shim-pack Scepter C18-120 (20 mm × 2.1 mm I.D., 1.9 µm)* 1 Temperature: 30 °C Injection Volume: 1 µL Mobile Phases: A) 0.05 % Formic acid in water B) 0.05 % Formic acid in acetonitrile Flowrate: 0.4 mL/min Time Program: B conc. 3 % (0.00 - 0.05 min) → 95 % (0.20 - 0.60 min) → 3 % (0.61-0.80 min) Rinsing Type: External rinse Mixer: Micro mixer Sample Loop Volume: 15 µL Vial: Shim-vial H glass* 2 (150 µL) MS Conditions System: LCMS-8060RX Ionization: ESI (Positive and negative mode) Mode: MRM Nebulizing Gas Flow: 5 L/min Heating Gas Flow: 15 L/min Interface Temp.: 400 °C DL Temp.: 300 °C Block Heater Temp.: 400 °C Drying Gas Flow: 5 L/min *1 P/N: 227-31012-01 *2 P/N: 227-34500-11 Compound Polarity Target m/z Reference m/z Resorufin + 214.10 > 186.10 214.10 > 103.10 4’-Hydroxydiclofenac + 312.00 > 230.10 312.00 > 265.90 Oxidized Nifedipine + 345.10 > 284.10 345.10 > 152.00 6-Hydroxychlorzoxazone - 184.00 > 64.05 184.00 > 120.00 7-Hydroxycoumarin - 161.00 > 133.10 161.00 > 105.10 Hydroxy Tolbutamide - 285.10 > 186.00 285.10 > 104.10 A Shim-vial H (150 μL) was used as the vial (Fig. 3). This small-volume vials with their unique shape are made of the same materials and receive the same cleaning treatment as the 1.5 mL vials. They suppress adsorption more than other small-volume inserts by limiting the contact surface between the sample and the container. In addition, the bottom surface of the vial is hollowed out in order to increase thermal conductivity. (Residual volume: Approx. 10 μL) Fig. 1 Nexera X4 and LCMS-8060RX Fig. 2 Pretreatment Procedure Table 2 Target Compounds and MRM Conditions Table 1 Nexera X4 and LCMS-8060RX Analysis Conditions Fig. 3 Shim-vial H (150 µL) Vial
Application News The LCMS-8060RX was used as the mass spectrometer. CoreSpray, adopted in the LCMS-8060RX, achieves stable ionization by further improving nebulizer flow uniformity and extending the upper flowrate limit to 7 L/min. Consistent ion injection reduces variation within the same sample and between instruments (Fig. 4). The Nexera X4 LC unit used, provides highly consistent solvent delivery during gradient analysis and demonstrates excellent retention time repeatability even in ultra-high-speed analysis. That significantly increases analysis speed while maintaining analytical quality equivalent to conventional HPLC. The analysis time for this method, including column equilibration, is 0.80 min, enabling rapid processing of a huge number of samples. Furthermore, the Nexera X4 features tool- less fittings (Fig. 5) for column connections, allowing simple and reliable column replacement even for ultra-high pressure analysis. Compound Retention time (min) Standard deviation of retention time (sec) Area %RSD Resorufin 0.399 0.010 2.54 4’-Hydroxydiclofenac 0.432 0.011 2.38 Oxidized Nifedipine 0.451 0.013 1.74 6-Hydroxychlorzoxazone 0.391 0.012 1.96 7-Hydroxycoumarin 0.399 0.010 1.65 Hydroxy Tolbutamide 0.400 0.012 2.28 Evaluation of Retention Time and Peak Area Repeatability A 10 nmol/L standard sample was analyzed six times using the indicated method to evaluate the repeatability of retention times and peak areas. The standard deviation of retention times was 0.010 to 0.013 sec, and the relative standard deviation of peak area was 3.0 % or less, confirming that sufficient analytical consistency was maintained even under high-speed gradient conditions. Results for Standard Samples Calibration standards were prepared over the range of 0.01 to 200 nmol/L, and linearity was confirmed from the results of six repeated analyses. The calibration range and coefficients of determination are summarized in Table 4, and representative chromatograms are shown in Fig. 6. The results showed good linearity for all compounds, with R 2 > 0.995 within the specified concentration range. All calibration points satisfied the criteria of accuracy within 80–120 % and precision within 20 % RSD for concentration. Compound Quantification Range (nmol/L) Coefficient of Determination R 2 Resorufin 0.5 – 100 0.999 4’-Hydroxydiclofenac 0.1 – 200 0.996 Oxidized Nifedipine 0.01 – 50 0.999 6-Hydroxychlorzoxazone 0.5 – 200 0.995 7-Hydroxycoumarin 0.5 – 100 0.995 Hydroxy Tolbutamide 0.05 – 200 0.997 8.91e4 Q 214.10>186.10 (+) 0.0 0.2 0.4 0.6 0.0e0 2.0e4 4.0e4 6.0e4 8.0e4 3.61e4 Q 184.00>64.05 (-) 0.2 0.4 0.6 0.0e0 1.0e4 2.0e4 3.0e4 2.60e5 Q 312.00>230.10 (+) 0.2 0.4 0.6 0.0e0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 7.86e4 Q 161.00>133.10 (-) 0.2 0.4 0.6 0.0e0 2.0e4 4.0e4 6.0e4 2.86e6 Q 345.10>284.10 (+) 0.2 0.4 0.6 0.0e0 5.0e5 1.0e6 1.5e6 2.0e6 2.5e6 5.09e5 Q 285.10>186.00 (-) 0.2 0.4 0.6 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 5.0e5 1.16e3 Q 312.00>230.10 (+) 0.2 0.4 0.6 0.0e0 2.0e3 4.0e3 6.0e3 7.78e2 Q 345.10>284.10 (+) 0.2 0.4 0.6 0.0e0 1.0e3 2.0e3 3.0e3 4.0e3 2.39e2 Q 161.00>133.10 (-) 0.2 0.4 0.6 0.0e0 2.0e2 4.0e2 6.0e2 8.0e2 1.04e6 Q 312.00>230.10 (+) 0.2 0.4 0.6 0.0e0 2.0e5 4.0e5 6.0e5 8.0e5 1.0e6 1.03e7 Q 345.10>284.10 (+) 0.2 0.4 0.6 0.0e0 2.0e6 4.0e6 6.0e6 8.0e6 1.0e7 6.01e5 Q 161.00>133.10 (-) 0.2 0.4 0.6 0.0e0 1.0e5 2.0e5 3.0e5 4.0e5 5.0e5 6.0e5 Resorufin 6-Hydroxychlorzoxazone Oxidized Nifedipine 4’-Hydroxydiclofenac 7-Hydroxycoumarin Hydroxy Tolbutamide Oxidized Nifedipine 4’-Hydroxydiclofenac 7-Hydroxycoumarin Unspiked Substrate-spiked Unspiked Substrate-spiked Unspiked Substrate-spicked Results for CYP-Metabolized Samples Using the same method, samples after CYP-mediated metabolic reactions were analyzed. As shown in the chromatograms in Fig. 7, no interfering peaks were observed in the unspiked sample solution, while the target metabolites (4’-hydroxydiclofenac, oxidized nifedipine, and 7-hydroxycoumarin) were detected with good peak shapes in the substrate-spiked sample solution. Fig. 4 CoreSpray Injection in the LCMS-8060RX Fig. 5 Tool-less Fittings in Nexera X4 System Table 3 Evaluation of Retention Time and Peak Area Repeatability (n = 6) Table 4 Calibration Summary Table Fig. 6 Representative Chromatograms (10 nmol/L) Fig. 7 Chromatograms of CYP-Metabolized Samples
Application News www.shimadzu.com/an/ Shimadzu Corporation © Shimadzu Corporation, 2026 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See https://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. First Edition: Aug. 2026 01-01138-EN Nexera, LCMS, Shim-pack Scepter, and Shim-vial are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries. Compound Spiked conc. MF (Average, n = 6) Conc. %RSD (n = 6) Resorufin 5 nmol/L 0.98 2.08 4’-Hydroxydiclofenac 0.90 2.98 Oxidized Nifedipine 0.89 2.98 6-Hydroxychlorzoxazone 1.07 4.03 7-Hydroxycoumarin 1.03 1.91 Hydroxy Tolbutamide 0.99 3.74 Evaluation of Matrix Effects The matrix factor (MF) was evaluated by adding the target metabolites to an unspiked CYP metabolic reaction sample solution. The results are indicated in Table 5. The MF ranged from 0.89 to 1.07, indicating no significant matrix effects and confirming that consistent analysis is possible with this method. Conclusion Using the Nexera X4 ultra high-performance liquid chromatograph and the LCMS-8060RX triple quadrupole mass spectrometer, six drug metabolites were analyzed. Despite ultra- fast 0.80 min analysis times, including column equilibration, retention time and peak area repeatability were good, confirming that consistent analysis is possible. This analytical system is expected to be useful for analyses that require high-speed performance, such as pharmacokinetic studies, where a large number of samples must be processed in a short time. Table 5 Results of Matrix Effect Evaluation
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