The RI1 is calculated by the following equation, where n is the number of carbon atoms of the calibrant used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound , tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant found on our column, we use the same equation to obtain the corresponding RI with the first two eluting DMED labeled fatty acid as the calibrants.
RI2: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 9 DMED labeled fatty acid standards with carbon chain length from C20 to C30, which is added into the targeted compounds to calibrate the retention time drifts to LC-MS analysis. The RI of a given compound was determined by measuring the retention time of this compound and linear regression equation of calibrants.
Previous studies have shown that the RTs of DMED-labeled FAs on C18 column increased with increasing carbon number of FAs (C20−C30). A linear regression equation (eq 1) can be obtained by analyzing the relationship of carbon number of calibrants and RT: y = b+kx, where y denotes RT, and x denotes carbon number of calibrants. The retention index for FAHFAs is defined as RI2= 100 × n. The RI2 of FAHFA is calculated according to the fitting equation-2 (eq 2) from the measured RT of the FAHFAs :
RI3: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use local linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 10 4-AMBA labeled fatty acid standards with carbon chain length from C1 to C10, which is added into the targeted compounds to calibrate the retention time. The RI of a given compound was determined by measuring the retention time of this compound and two calibrants whose retention time neighbors this compound.
The RI3 is calculated by the following equation, where n is the number of carbon atoms of the calibrant used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound, tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant (4-AMBA labeled formic acid), we use the same equation to obtain the corresponding RI with selecting the 4-AMBA labeled formic acid and acetic acid as the calibrants.
RI4: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use local linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 10 CAPA a labeled fatty acid standards with carbon chain length from C1 to C10, which is added into the targeted compounds to calibrate the retention time. The RI of a given compound was determined by measuring the retention time of this compound and two calibrants whose retention time neighbors this compound.
The RI4 is calculated by the following equation, where n is the number of carbon atoms of the calibrant used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound, tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant (CAPA a labeled formic acid), we use the same equation to obtain the corresponding RI with selecting the CAPA a labeled formic acid and acetic acid as the calibrants.
RI5: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use local linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 16 DMAP labeled fatty amine standards with carbon chain length from C1 to C16. The calibrants are mixed with the targeted compounds before LC-MS analysis, to calibrate the retention time. The RI of a given compound is determined by measuring the retention time of this compound and two calibrants whose retention time neighbors this compound.
The RI5 is calculated by the following equation, where n is the number of carbon atoms in carbon chain of the calibrants used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound, tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant (DMAP labeled methylamine), we use the same equation to obtain the corresponding RI with selecting the DMAP labeled methylamine and ethylamine as the calibrants. If a compound is eluted after the last calibrant (DMAP labeled cetylamine), we selected DMAP labeled pentadecanamine and cetylamine as the calibrants to obtain the corresponding RI.
RI6: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use local linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 16 DMPI labeled fatty amine standards with carbon chain length from C1 to C16. The calibrants are mixed with the targeted compounds before LC-MS analysis, to calibrate the retention time. The RI of a given compound is determined by measuring the retention time of this compound and two calibrants whose retention time neighbors this compound.
The RI6 is calculated by the following equation, where n is the number of carbon atoms in carbon chain of the calibrants used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound, tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant (DMPI labeled methylamine), we use the same equation to obtain the corresponding RI with selecting the DMPI labeled methylamine and ethylamine as the calibrants. If a compound is eluted after the last calibrant (DMPI labeled cetylamine), we selected DMPI labeled pentadecanamine and cetylamine as the calibrants to obtain the corresponding RI.
RI7: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use local linear regression to calibrate the chromatographic retention time drifts of targeted compounds on C18 column with calibrants. The calibrants is a mixture of 20 MPEA labeled fatty acid standards with carbon chain length from C1 to C20, which is added into the targeted compounds to calibrate the retention time. The RI of a given compound was determined by measuring the retention time of this compound and two calibrants whose retention time neighbors this compound.
The RI7 is calculated by the following equation, where n is the number of carbon atoms of the calibrant used for calibration whose retention time is before the compound and n+1 is that after the compound. And tcompound, tn and tn+1 is the retention time of the given compound and two calibrants. If a compound is eluted before the first calibrant (MPEA labeled formic acid), we use the same equation to obtain the corresponding RI with selecting the MPEA labeled formic acid and acetic acid as the calibrants.
RI8: In our chemically labeled-based HPLC retention Index (CL-HPLC RI) model, we use linear regression to calibrate the chromatographic retention time drifts of targeted compounds on HILIC column with calibrants. The calibrants is a mixture of 23 DMED labeled fatty acid standards with carbon chain length from C2 to C24, which is added into the targeted compounds to calibrate the retention time. The RI of a given compound was determined by measuring the retention time of this compound and linear regression equation of calibrants.
Previous studies have shown that the RTs of DMED-labeled FAs on HILIC column decreased with increasing carbon number of FAs (C2−C24). A linear regression equation (eq 1) can be obtained by analyzing the relationship of carbon number of calibrants and RT: y = b+ax, where y denotes log10.
