Quantitative measurement of BMAA, DAB and AEG as AQC derivatives was performed using RPLC-MS/MS on an Agilent 1200 LC system (Agilent Inc., Palo Alta, CA) connected to an API4000 QTRAP mass spectrometer (AB Sciex, Concord, ON, Canada) using electrospray ionization and selected reaction monitoring. Direct quantitation of BMAA, DAB and AEG by HILIC-MS/MS on an Agilent 1100 LC system connected to an API 4000 mass spectrometer. Table 1 provides the experimental details for the two methods.
Api 4000 mass spectrometer
Manufactured by Agilent Technologies
The API 4000 mass spectrometer is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) system designed for analytical laboratories. It offers accurate and sensitive quantitative analysis of a wide range of analytes. The core function of the API 4000 is to separate, detect, and measure the mass-to-charge ratio of ions in a sample, providing detailed information about the chemical composition of the analyzed material.
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3 protocols using api 4000 mass spectrometer
1
Quantitative measurement of AQC derivatives
2
Quantifying Phytohormones in Maize Leaves
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Maize leaves samples (approximately 100 mg) were harvested and transferred to FastPrep tubes. One milliliter of ethyl acetate spiked with 200 ng of d5-JA, d6-ABA, and d4-SA was added to each sample (n = 9) and used as the internal standards for JA, ABA, and SA, respectively. Ten-microlitre aliquots of the samples were analyzed using a triple quadrupole liquid chromatography with tandem mass spectrometry (LC−MS/MS) system (Shimadzu LC-20A coupled with an Applied Biosystems API4000 mass spectrometer); this apparatus was equipped with an SB-C18 column (2.1 mm×150 mm, 3.5 µm; Agilent Technologies) and kept in a thermostat-controlled chamber at 35°C. A mobile phase composed of solvent A (0.1% formic acid) and solvent B (acetonitrile) was used in gradient mode for separation at a constant flow rate of 0.2 mL min−1. The compounds were detected in electrospray ionization negative mode. JA, SA, and ABA were quantified by comparing their peak areas with the peak areas of their respective internal standards (Pan et al., 2021 (link)).
3
Quantification of IMU-838 in Plasma and Urine
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All samples were stored at − 20 °C until they were analyzed. Plasma and urine IMU-838 concentrations were analyzed using a validated liquid chromatography with a tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS system consists of an Applied Biosystems API 4000 mass spectrometer, chromatographic system of an Agilent 1200 series G1312A/B binary pump, Agilent 1200 series G1322/A degasser and Agilent 1200 series G1316A column oven, and an HTC PAL autosampler CTC analytics. The assay method involved a protein precipitation of samples with the internal standard (SC-13093), a chromatographic separation on a Luna C8 (50 × 2.0 mm, 3 μm) column and LC–MS/MS detection. The lower limit of quantitation for the assay was 0.100 µg/ml, and the linear calibration range was 0.100–25.0 µg/ml. The mobile phase used was: mobile phase A [0.7708 g ammonium acetate + 1000 ml high-performance liquid chromatography (HPLC)-water adjust to pH 5 with acetic acid] and mobile phase B (acetonitrile). The purge solution was: purge solution 1 (500 μl formic acid and 500 ml HPLC-water) and purge solution 2 (200 ml HPLC-water, 300 ml acetonitrile and 1.00 ml ammonia solution). The electrospray (atmospheric pressure ionization) technique was used in negative ion (ESI−) mode. The injection volume was 20.0 μl (in the SAD study) and 5.0–10.0 μl (in the MAD study), and the flow rate was 0.300 ml/min.
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