A simple liquid chromatography tandem mass spectrometric method for fast detection of hydrogen sulfide based on thiolysis of 7-nitro-2, 1, 3-benzoxadiazole ether.

The long identified toxic gas, hydrogen sulfide (H2S), which has also been confirmed as the third gaseous signaling molecule following NO and CO, plays important roles in various physiological and pathological process. The current most established quantification method for H2S is HPLC method coupled with fluorescence detection after derivatization with a costly fluorescent reagent, Monobromobimane (MBB). However, The MBB method is characterized by strict reaction condition, long reaction time, tedious operation, and inconsistent reported results. In this study, based on the thiolysis reaction of 7-nitro-2, 1, 3-benzoxadiazole (NBD) ether, the commonly used chromatographic modifier 4-chloro-7-nitro-2,1,3- benzoxadiazole (NBDCl) and four probes (NBDOMe, NBDOEt, NBDOTFE and NBDOCMR) synthesized from NBDCl were tested as alternatives for fast quantification of H2S by LC-MS/MS. The reaction product between NBD ethers/NBDCl and H2S showed special pink color visible to the naked eye and was easy to synthesize and separate in lab; it also showed good retention on common chromatographic columns and high instrument response; therefore it is a good determinand. After establishment of LC-MS/MS methods for all the related compounds, the reaction conditions were optimized for all the probes with H2S. Then the stability, selectivity, reaction rate, sensitivity and quantitative linear relationship between the reaction product and H2S concentration were studied for each probe. Finally, NBDOEt was selected for LC-MS/MS detection of H2S. In comparision with the MBB method, the established NBDOEt method showed matched sensitivity and linearity, better selectivity, and higher repeatability; and had the advantages of easy operation, simple reaction condition, and cheap raw materials. The method was successfully validated and applied to determination of Na2S content in Na2S∙9H2O bulk drug and injection. In conclusion, NBDOEt is a promising option for quantification of H2S in abiotic matrix.

Fast and direct determination of catechol-3, 6-bis(methyleiminodiacetic acid) prototype in beagle dog plasma using liquid chromatography tandem mass spectrometry: A simplified and high throughput in-vivo method for the metal chelator.

It is usually somewhat difficult to analyze the metal chelators, especially in complex biological matrix, because of the interference of metal ions in both the matrix and analyzing system. In this study, an innovative and simple bioanalytical method was established and validated for the quantification of a newly developed uranium chelator catechol-3, 6-bis (methyleiminodiacetic acid) (CBMIDA) in beagle dog plasma. Different analytical columns and mobile phase were tested for effective chromatography resolution and sensitive and reproducible response of CBMIDA and the internal standard. An Agilent Zorbax SB AQ column was chosen. Excessive peak tailing, peak asymmetry, low recovery, and poor reproducibility, which are generally observed in chromatographic analysis of metal chelators, were overcome by the use of a pulse gradient method and addition of ethylene diamine tetraacetic acid (EDTA) to the mobile phase at 8 µg mL-1, enabling good peak shape, low matrix interference, high precision and good linearity for CBMIDA quantification in beagle dog plasma. Plasma sample pretreatment was performed by a simple, high throughput protein precipitation step with 2.5 mM EDTA methanol solution in a 96-well protein precipitation plate without complexing with the metal ions, and the sample was directly analyzed by electrospray ionization mass spectrometry. By shifting the analysis target from the metal complex to metal chelator itself, the method has an advantage over the existing method for determination of EDTA and diethylenetriaminepentaacetic acid owing to increased sample throughput and apparent simplicity. The assay was validated in accordance with the United States Food and Drug Administration guidelines and successfully applied to the pharmacokinetic study of CBMIDA in beagles after intramuscular injection of CBMIDA at different doses. The method was sensitive enough for the detection of CBMIDA concentration at 4 elimination half-times. The experimental strategies presented herein may be helpful for the measurement of other radionuclide chelators in biological matrices.

Simultaneous determination of the potent anti-tuberculosis regimen-Pyrazinamide, ethambutol, protionamide, clofazimine in beagle dog plasma using LC-MS/MS method coupled with 96-well format plate.

Tuberculosis is one of the top concerns in the world and acutely threatens human health. A new potent candidate regimen containing pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO) and clofazimine (CFZ) was proposed by Parabolic Response Surface/Feedback System Control (FSC/PRS) system and showed excellent outcomes in vitro and vivo studies. Here, a convenient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneously determination of four compounds in beagle dog plasma. The plasma samples, 50 µL for each, were pretreated by methanol on 96-well format plates and a further dilution step was designed to reduce predictable matrix effect and lessen the burden of subsequent analysis. The chromatographic separation was achieved on an Agilent SB-Aq column (4.6 mm × 150 mm, 5 µm) at 30 °C by a gradient elution within 6 min. The mobile phase was a mixture of 0.2% formic acid-5 mM ammonium acetate aqueous solution (phase A) and 0.2% formic acid methanol (phase B) with a total flow rate of 1 mL/min. The 30% of post-column eluant was injected into mass spectrometer, equipped with electrospray ionization (ESI) source under positive mode and multiple-reaction monitoring (MRM). This quantification method was proved to be satisfied in selectivity, accuracy, precision, linearity (r2 > 0.998), recovery, matrix effect and stability. Under the specialized conditions, the calibration curves ranged from 20 to 5000 ng/mL for PZA, 1 to 500 ng/mL for EMB, 1 to 500 ng/mL for PTO, and 1 to 200 ng/mL for CFZ. The quantitative accuracy was further assessed under different degrees of hemolyses in detail. This method was proved to be robust and efficient, and successfully applied to the pharmacokinetic study of the new regimen in Beagle dogs.