A quantification method for trace level of oxytocin in food matrices using LC-MS/MS

Abstract

Backgrounds: Oxytocin is nowadays used to increase the agricultural products besides its use during the milking of cattle leading to the contamination of agricultural produce and milk with oxytocin. Monitoring of accurate oxytocin contaminations from foodstuffs is sometimes required to maintain the quality standard. The commonly used oxytocin assays in this study were interfered with by the food matrix. There is a need to develop an accurate and confirmed method for monitoring oxytocin contaminations in foodstuffs.Objective: An attempt is made to develop an accurate assay method of oxytocin from milk and agricultural produces.Methods: The acidified methanol was used for the extraction of oxytocin from target food stuff/matrices (agricultural produce and Milk). LC-MS/MS was used for its detection and quantification. In the chromatographic separation, Oxytocin concentration was optimized using selective reaction monitoring (SRM) with heated electrospray ionization (HESI) in positive polarity. The chromatographic separation was performed using a reversed-phase C18 column with gradient elution at a flow rate of 0.4 ml/min. The acidified methanol was used for the extraction of oxytocin in all target food matrices. The method performance was verified as per the SANTE 2021 guideline. After method validation, the method was applied in real food samples analysis for assessing the presence/absence of oxytocin.Results: The calibration curve offered excellent linearity (R2 = 0.999) with less than 15% residuals. The matrix effect was <20% observed for all target matrices. The mean recoveries were within 70%–115% with <11% RSD at four different levels in milk and 0.01 mg/kg in fruits and vegetables. The optimized method was applied to 50 random samples of milk, fruits, and vegetables from the market for the purposes of an established quality control approach. Based on the results, we did observe a signal of oxytocin in the random samples Therefore, this method has shown its practical suitability for the detection of oxytocin in milk, fruits, and vegetables.Conclusion: Extraction of oxytocin using acidified methanol followed by assays using LC-MS/MS is a simple, sensitive, accurate, reproducible, and practically suitable method for detection and quantification of oxytocin from milk, fruits, and vegetables.

Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers

“…Previous studies have also reported similar observations, highlighting the potential of hydrophilic and rigid spacers to improve assay sensitivity and specificity. It has also been previously reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA ( 1 – 5 , 11 , 19 , 20 ) as it does have an effect in some other immunoassays and auxiliary binding systems ( 9 – 11 , 20 ). The use of a hydrophilic and rigid spacer possibly helped in projecting the hapten away from the enzyme, leading to an enhanced antibody binding signal and improved sensitivity of the assay ( 9 – 11 , 14 , 20 ).…”

“…The concentration was plotted on the X-axis (log scale) and the A/A0x100 on the Y-axis. The values of the unknown samples were calculated by an in-house–developed personal computer program written in QBASIC language using the logit–log linear regression method according to the method described elsewhere ( 5 ). The affinity constant of the PSL antibody for PSL was estimated by a Scatchard plot according to the method described elsewhere ( 10 ).…”

“…The study revealed that the use of a spacer between the steroid and enzyme in the enzyme conjugate increases the sensitivity and specificity of the assay. It has also been reported that the spacer length does not bear any correlation with the assay sensitivity in an ELISA of steroids ( 1 , 5 – 11 , 14 , 19 , 20 ). Further, the nature of the spacer (hydrophilic, hydrophobic, flexible, or rigid) is related to assay sensitivity and not to spacer length ( 5 – 11 , 14 , 33 ).…”

“…It has also been reported that the spacer length does not bear any correlation with the assay sensitivity in an ELISA of steroids ( 1 , 5 – 11 , 14 , 19 , 20 ). Further, the nature of the spacer (hydrophilic, hydrophobic, flexible, or rigid) is related to assay sensitivity and not to spacer length ( 5 – 11 , 14 , 33 ). This disparity in the behavior of spacers towards the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between…”

“…In the antibody-immobilized format, spacers have been coupled between steroid derivative and label protein to minimize the orthodox bridge recognition effects and to overcome steric constraints between the two high molecular weight proteins, i.e., the antibody and label ( 5 – 11 , 14 , 19 , 20 ). Shrivastav et al.…”