I am back here to complete my Amphetamines blog series. In my previous posts, I have discussed the importance of separating the chiral d- and -l isomers to accurately identify the illicit isomer using an achiral method on a Raptor C18 column employing a pre-column derivatization technique, sample prep strategies and derivatization efficiency in my previous blogs part I, part II & part III . Today I’d like to discuss more about the validation studies by evaluating the method’s accuracy, precision, selectivity and specificity.
Once the method development was completed, I wanted to perform some additional work like validation studies to ensure that this technique is suitable to quantify the isomers even in the human urine matrix using deuterated internal standards for both d-and-l amphetamines and methamphetamines. The internal standards were also derivatized simultaneously with the analytes in urine matrix from 50-5000ng/mL calibration range along with the QC standards. As this sample derivatization is simple, fast and reproducible with maximum derivatization efficiency it was easy to prepare large number of samples at a time for validation studies.
The validation experiments were performed as described below:
Linearity: For linearity experiments, I have injected the calibrators in the range of 50-5000ng/mL followed by 3 sets of QC standards. Using 1/x weighted linear regression, all four analytes showed acceptable linearity with r2 values of 0.998 or greater (Figure 1). In addition, the %deviation from nominal concentration was concentration was <15%, in all 3 accuracy and precision experiments (Table I)
Figure 1: Standard Curves
Accuracy and Precision: Once a suitable linearity was achieved, precision and accuracy analyses were performed on three different days by injecting the 3 sets of calibration standards followed by 3 sets QC standards at 4 levels (LLOQ, LQC, MQC and HQC) prepared in pooled urine in 3 different batches. Method accuracy was demonstrated by the average recovery values within 10% of the nominal concentrations for low, mid, and high QC levels and within 15% for the LLOQ. The %RSD was 1–8% and 0.6–8% for intraday and interday results, respectively, indicating acceptable method precision (Table I). All the calculations were performed by averaging the data from 3 different days, except intra-day studies. Because deuterated internal standards were used for each enantiomer, the standards and target analytes experienced similar enhancements, which ensured accurate and reliable quantitative results were obtained.
Table I: Interday Accuracy and Precision studies for the analysis of amphetamines by LC-MS/MS in urine
Selectivity and Specificity: As we know when we have an analyte in high concentration eluting closely with low levels of another compound the chromatographic resolution between these compounds is compromised, making it difficult for true identification of the illicit methamphetamine consumption. This is very important especially with the separation of isobaric chiral compounds at extreme levels of each other (very high of d-isomer and very low of l-isomer, vice versa). We were very curious to see how a real urine sample after consumption of the illegal methamphetamine with high intense d- isomer looks like!!! And similarly, consumption of over-the-counter (OTC) drugs that contain l-methamphetamine can result in a high-intensity l-enantiomer peak in urine, which may make it difficult to identify very low concentrations of the illegal enantiomer (d-methamphetamine), leading to false negative results.
Well, to address our curiosity and the method’s specificity, here we performed an experiment by spiking very high levels of the d- isomer with LLOQ levels of l- isomers and vice versa and analyzed. The method developed here was found to be highly specific and selective with good chiral resolution even when evaluated at extreme concentrations, such as high l- enantiomer (5000 ng/mL) with low d- enantiomer (50 ng/mL) for both amphetamines and methamphetamines in urine (Figure 2). The intense signal from d-isomer didn't negatively impact the chromatographic resolution and peak shape of low levels of l-isomer.
Figure 2: Highly selective chromatographic results were obtained even at extreme concentrations for both the d- and l-enantiomers.
I hope this blog was helpful for your sample prep and method development for achiral separation of chiral amphetamines using a Raptor C18 column. If you would like more information on this work click on the following link for more details on the full application note and the references: Analysis of Amphetamines by LC-MS/MS for High-Throughput Urine Drug Testing Labs
1. Newmeyer, N. M, Concheiro, M and Huestis, A. M. J Chromatogr A. 2014; 1358: 68–74. 2. Foster, S. B and Gilbert, D. D. J Analytical Toxicology.1998; 22:265-9.
Links to blogs in this series: