The following criteria are guidelines based on recommendations by the U.S. Department of Health and Human Services Food and Drug Administration for standardizing industry bioanalytical method validation. Full method validation may not be necessary for collaborators of the UNC Chemistry MS Core, but it is important to consider the following questions:

• Does the method measure the intended analyte? For example, does anything interfere with the measurement, and is the method specific or selective for the analyte?
• What is the variability associated with these measurements? For example, what are the accuracy and precision of the method?
• What is the range in measurements that provide reliable data? For example, what are the lower and upper limits of quantification of the method?
• How do sample collection, handling, and storage affect the reliability of the data from the method? For example, what steps need to be followed while collecting samples? Do the samples need to be frozen during shipping? What temperatures are required to store the samples, and how long can samples be stored?

Before development of a method, the collaborator should understand the physicochemical properties of the analyte of interest or communicate with the UNC Chemistry MS Core employees to identify the analyte properties that may affect the quantitative method.

Method development involves optimizing the procedures and conditions involved with extracting and detecting the analyte.

a. Reference Standards

Authenticated analytical reference standards (when available) with known identities and purities should be used to prepare solutions of known concentrations.

Stable, isotopically-labeled internal standards (IS) should be added to every sample (calibrator, quality control, unknown) prior to extraction from the biological matrix to correct for loss of analyte during sample preparation, chromatographic separation, and ionization for mass spectrometric (MS) analysis. The heavy-isotope labeled IS should have a structure identical to the analyte, but contain 3 or more heavy atoms (e.g. ²H, ¹³C, ¹⁵N). The analyte and IS should co-elute from the liquid chromatography (LC) column, therefore undergoing co-ionization prior to MS. The heavy-isotope labels allow differentiation of analyte and IS by mass-to-charge ratio.

b.  Calibration Curve

The preferred calibration technique is external calibration with internal standardization. For the greatest method accuracy, external calibration standards should be prepared in the same biological matrix as the unknown samples and exposed to the same sample processing protocol as unknown samples. If a blank biological matrix (without endogenous analyte or interference) is not available, the standard addition method may be used (if unknown sample volume/amount is sufficient) or external calibrators may be prepared in neat solvent (at the risk of decreased method accuracy).

Each calibration curve should contain a matrix blank (no analyte, no IS), a zero calibrator (matrix blank plus IS), and a minimum of six non-zero calibrators that cover the relevant concentration range. Non-zero calibrators’ response should be within 15% of nominal concentrations, except at the LLOQ which should be within 20% of the nominal concentration. A calibration curve can be accepted when 75% of the calibrators meet these criteria.

c. Quality Control Samples

Quality control (QC) samples assess the precision and accuracy of the method. QCs should be prepared in the same matrix as unknown samples and calibrators, but using a different stock solution from the calibrators. QCs should be prepared at low, mid and high concentrations relative to the calibration range. Acceptance criteria require ≥67% of total QCs to be within 20% of nominal concentration, and ≥50% of QCs per concentration level should be within 20% of their nominal.

d. Selectivity and Specificity

Ensuring selectivity and specificity means to confirm that the substance being measured is the intended analyte and to assess the level of chemical interference. A study may be designed to assess the effects of the matrix on ion suppression, ion enhancement, or extraction efficiency. This matrix effect and extraction recovery study includes processing and analyzing blank matrix samples, spiked matrix samples, post-extraction spiked matrix samples and spiked solvent samples.

e. Accuracy, Precision, Recovery and Stability

If a full method validation is required the accuracy and precision of the method will be assessed across multiple days. Three intra-day sets of replicate QCs and calibrators will be analyzed on three different days (inter-day). Therefore, 9 sets of QC and calibrator replicates will be analyzed [ 3 (intra-day) x 3 (inter-day) ].

For full method validation the stability of the analyte in a given matrix across many scenarios must be assessed. For details on both the accuracy and precision and the stability studies refer to the FDA’s Bioanalytical Method Validation Guidance for Industry.

f. Sample Analysis Sequence and Carryover

Solvent blanks are injected periodically to minimize, and confirm the absence of, carryover. At least two solvent blanks must precede the analytical run, followed by a zero calibrator (blank + IS) and the calibration curve from low to high concentration. Solvent blanks must be injected after the highest concentration calibrator, prior to QCs. Solvent blanks must always precede and succeed both the calibration curve and QCs. Unknown samples are then injected, with interspersed solvent blanks and QC samples (if there are many unknown samples). An analytical run will always conclude with QCs and a reanalysis of the calibration curve.

Sample sequence: Solvent Blanks > Calibrators > Solvent Blanks > QCs > Solvent Blanks > Unknown Samples (with interspersed Solvent Blanks) > Solvent Blanks > QCs > Solvent Blanks > Calibration Curve > Solvent Blanks

Criteria to Confirm the Detection and Quantification of an Analyte by LC-MS/MS

1. One quantifying, and two qualifying product ions (MS/MS transitions) must be detected in the sample.
2. There are no qualifying or quantifying product ions detected in blank matrix at the analyte LC retention time.
3. Mean of product ion ratio of unknown samples is in the range of ±20% of mean of product ion ratio of calibrator/QC samples.
4. Mean of retention times of the unknown samples is in the range of ±0.2 min of mean of calibrator/QC samples.
5. The ratio of chromatographic peak areas of the quantifying analyte product ion response and of the internal standard product ion response is used for calibration and quantification.
6. Linear regression is employed for quantification.
7. Weighting factor may be used for linear regression.
8. Concentrations of the LLOQ calibrator should be within ±20% of the nominal value and the concentrations of other than LLOQ calibrators should be within 15% of the nominal value.
9. Exclusion of calibrators is acceptable but at least 75% of the calibrators, with a minimum of six calibration standard levels, must fulfil above criterion. If one calibrator is excluded, the calibration curve without this calibration standard should be re-evaluated, and regression analysis performed.
10. The calibration curve should be prepared using freshly spiked samples, it is not allowed to use previously prepared and stored calibration samples.
11. The mean concentration of QC samples should be within 20 % of the nominal values. At least 67% of the QC samples and at least 50% at each concentration level should comply with this criterion.
12. The conditions above must be met to accept the analytical run.