Multiplexed external calibrator and control for screening and diagnostic assays

A method of quantifying a target analyte in a series of samples by mass spectrometry, the method comprising:

• obtaining a mass spectrometer signal for a calibration mixture, wherein the calibration mixture comprises a plurality of calibrants comprising at least a standard of the target analyte reacted with a first derivatizing agent, the target analyte reacted with a second derivatizing agent, and the target analyte reacted with a third derivatizing agent, the first, second and third derivatizing agents being isotopic analogues of one another and wherein said plurality of calibrants are distinguishable by mass spectrometry from one another and have different, known concentrations from one another in said calibration mixture;
• adding an internal standard to the calibration mixture and each of said series of samples prior to obtaining the mass spectrometer signal for the calibration mixture and each of the series of samples, wherein the internal standard comprises the target analyte reacted with a fourth derivatizing agent comprising a fourth isotopic analog of the first, second, and third derivatizing agents distinguishable by mass spectrometry from the first derivatizing agent, the second derivatizing agent, and the third derivatizing agent, with the internal standard of the target analyte having a fourth concentration different than the respective concentrations of the plurality of calibrants, wherein each of the first, second, third, and fourth derivatizing agents has a substantially same physiochemical properties, with at least one atom in each of the first, second, third, and fourth derivatizing agents being a different isotope of the corresponding at least one atom of the other derivatizing agents;
• identifying from the mass spectrometer signal for the calibration mixture a characteristic response of each of said plurality of calibrants, wherein each of the characteristic responses is associated with a different m/z value corresponding to a different one of the plurality of calibrants and being indicative of the concentration of each of the plurality of calibrants in the calibration mixture;
• generating a single calibration curve for the calibration mixture based on said characteristic responses of the standard of the target analyte reacted with the first derivatizing agent, the target analyte reacted with the second derivatizing agent, the target analyte reacted with the third derivatizing agent, and the internal standard comprising target analyte reacted with the fourth derivatizing agent;
• creating a derivatized sample for each of the series of samples by reacting each of the series of samples with one of the first derivatizing agent, the second derivatizing agent, the third derivatizing agent or an isotopic analog thereof;
• obtaining a mass spectrometer signal from each of the derivatized samples from the series of samples;
• identifying from the mass spectrometer signal for each of the derivatized samples from the series of samples a characteristic response indicative of an unknown concentration of the target analyte in each derivatized sample, and a characteristic response indicative of the known fourth concentration of the internal standard comprising the target analyte reacted with the fourth derivatizing agent;
• and
• comparing the characteristic response of each of the derivatized samples in the series of samples to the single calibration curve to quantify the unknown concentration of the target analyte in each sample.

A method of quantifying an analyte in a series of samples by mass spectrometry, the method comprising:

• obtaining a mass spectrometer signal for a calibration mixture, wherein the calibration mixture comprises a plurality of calibrants comprising at least a first compound at a known target concentration in the calibration mixture, a first isotopic analog of the first compound at a known first concentration, and a second isotopic analog of the first compound at a known second concentration, wherein each of the first compound and the first and second isotopic analogs of the first compound are distinguishable by mass spectrometry from one another and have different, known concentrations from one another in said calibration mixture,
• adding an internal standard to the calibration mixture and each of said series of samples prior to obtaining the mass spectrometer signal for the calibration mixture and each of the series of samples, wherein the internal standard comprises a third isotopic analog of the first compound at a third known concentration distinguishable by mass spectrometry from the first compound, the first isotopic analog, and the second isotopic analog, wherein each of the first, second, and third isotopic analogs of the first compound has a substantially same physiochemical properties as the first compound, with at least one atom in each of the first, second, and third isotopic analogs being a different isotope of the corresponding at least one atom of the other isotopic analogs and of the standard of the first compound;
• identifying from the mass spectrometer signal for the calibration mixture a characteristic response of each of said plurality of calibrants, each of the characteristic responses is associated with a different m/z value corresponding to a different one of the plurality of calibrants and being indicative of the concentration of each of the plurality of calibrants in the calibration mixture;
• generating a single calibration curve for the calibration mixture based on said characteristic responses of the at least the first compound, the first isotopic analog of the first compound, the second isotopic analog of the first compound, and the internal standard comprising the third isotopic analog of the first compound;
• obtaining a mass spectrometer signal from each of the series of samples, each of the series of samples containing a target analyte having the same structure as the first compound or an isotopic analog of the first compound;
• identifying from the mass spectrometer signal for each sample a first compound characteristic response indicative of an unknown concentration of the target analyte in the respective each sample, and a characteristic response indicative of the known third concentration of the internal standard comprising the third isotopic analog of the first compound added to the respective sample;
• and
• comparing the characteristic response of the target analyte in each sample to the single calibration curve to quantify the unknown concentration of the target analyte in the respective each sample.