New Technology in LCMS

As with any scientific endeavor, the field of LC-MS is continuing to evolve. New instrumentation and new technology provide new opportunities for increasing sample throughput or providing improved data quality. In this section, I will discuss some of these new technology advances and briefly state how they might be used for discovery DMPK applications. The types of advances that are described include those in either chromatography or mass spectrometry.

One of the more exciting new advances in the chromatography field is ultraperformance liquid chromatography (UPLC) [130, 131]. UPLC is a system where higher (than HPLC) pressure can be used, which allows one to use smaller (<2 mm) particles in the column with a resulting improvement in the chromatographic resolution. UPLC was introduced as a commercial product recently and was quickly adapted for use as part of the LC-MS field. Several recent reports on the utility of UPLC-MS/MS have been published [132-137] in the literature. For example, Castro-Perez et al. [136] describe the utility of UPLC-MS/MS for the analysis of in vitro drug metabolism samples; in their examples, it is clear that

Table 13.1 Rules for discovery (nonGLP) "screen" assays (level I). Adapted from [6], with permission from Taylor and Francis Group.

1 Samples should be assayed using HPLC-MS/MS technology.

2 Sample preparation should consist of protein precipitation using an appropriate internal standard (IS).

3 Samples should be assayed along with a standard curve in duplicate (at the beginning and end of the sample set).

4 The zero standard is prepared and assayed, but is not included in the calibration curve regression.

5 Standard curve stock solutions are prepared after correcting the standard for the salt factor.

6 The standard curve should be three levels, typically ranging over 25-2500 ng mL_1 (they can be lower or higher as needed for the program). Each standard is 10x the one below (thus, a typical set would be 25, 250, 2500 ng mL_1). The matrix of the calibration curve should be from the same animal species and matrix type as the samples.

7 QC samples are not used and the assay is not validated.

8 After the assay, the proper standard curve range for the samples is selected. This must include only two concentrations in the range that covers the samples. A single order of magnitude range is preferred, but two orders of magnitude is acceptable, if needed to cover the samples.

9 Once the range is selected, at least three of the four assayed standards in the range must be included in the regression analysis. Regression is performed using unweighted linear regression (not forced through zero).

10 All standards included in the regression set must be back-calculated to within 27.5% of their nominal values.

11 The limit of quantitation (LOQ) may be set as either the lowest standard in the selected range or as 0.4 x the lowest standard in the selected range, but the LOQ must be greater than three times the mean value for the back-calculated value of the two zero (0) standards.

12 Samples below the LOQ are reported as zero (0).

13 If the LOQ is 0.4x the lowest standard in the selected range, then samples with back-calculated values between the LOQ and the lowest standard in the selected range may be reported as their calculated value, provided the S/N for the analyte is at least three (3).

14 Samples with back-calculated values between 1.0x and 2.0x the highest standard in the selected range are reportable by extending the calibration line up to 2x the high standard.

15 Samples found to have analyte concentrations more than 2x the highest standard in the regression set are not reportable. These samples must be reassayed after dilution or along with a standard curve that has higher concentrations so that the sample is within 2x the highest standard.

Table 13.2 Rules for discovery (nonGLP) ''full PK'' assays ( level II). Adapted from [6], with permission from Taylor and Francis Group.

1 Samples should be assayed using HPLC-MS/MS technology.

2 Sample preparation should consist of protein precipitation using an appropriate internal standard (IS).

3 Samples should be assayed along with a standard curve in duplicate (at the beginning and end of the sample set).

4 The zero standard is prepared and assayed, but is not included in the calibration curve regression.

5 Standard curve stock solutions are prepared after correcting the standard for the salt factor.

6 The standard curve should be 10-15 levels, typically ranging from 1 to 5000 or 10000 ng mL_1 (or higher as needed). The matrix of the calibration curve should be from the same animal species and matrix type as the samples.

7 QC samples are not used.

8 After the assay, the proper standard curve range for the samples is selected; this must include at least five (consecutive) concentrations.

9 Once the range is selected, at least 75% of the assayed standards in the range must be included in the regression analysis.

10 Regression can be performed using weighted or unweighted linear or smooth curve fitting (e.g., power curve or quadratic), but is not forced through zero.

11 All standards included in the regression set must be back calculated to within 27.5% of their nominal values.

12 The regression r2 must be 0.94 or larger.

13 The limit of quantitation (LOQ) may be set as either the lowest standard in the selected range or as 0.4x the lowest standard in the selected range, but the LOQ must be greater than three times the mean value for the back-calculated value of the two zero (0) standards.

14 Samples below the LOQ are reported as zero (0).

15 If the LOQ is 0.4 x the lowest standard in the selected range, then samples with back-calculated values between the LOQ and the lowest standard in the selected range may be reported as their calculated value provided the S/N for the analyte is at least three (3).

16 Samples with back-calculated values between 1.0 x and 2.0 x the highest standard in the selected range are reportable by extending the calibration curve up to 2x the high standard as long as the calibration curve regression was not performed using quadratic regression.

17 Samples found to have analyte concentrations more than 2x the highest standard in the regression set are not reportable. These samples must be reassayed after dilution or along with a standard curve that has higher concentrations so that the sample is within 2x the highest standard.

418 | 13 Mass Spectrometry in Early Pharmacokinetic Investigations Table 13.2 (continued)

18 The assay is not validated.

19 The final data does not need to have quality assurance (QA) approval. This is an exploratory, nonGLP study.

the improved chromatographic resolution provided by the UPLC system allows the scientist to obtain better data from the sample analysis.

Another recent innovation is the QTrap mass spectrometer. The QTrap MS system combines the capabilities of a triple quadrupole mass spectrometer and a linear ion trap mass spectrometer into one MS system. Initially, the QTrap MS was used primarily as a tool for metabolite identification studies [34, 35, 38]. As reported by Li et al. [138], the QTrap MS can also be used as an excellent system for the quantitative analysis of discovery PK samples. The advantage of the QTrap MS system for quantitative analysis is that it can be used to look for plasma metabolites of the NCE and provide an easy way to monitor them while providing the quantitative data on the NCE.

Two more MS-related innovations are worth noting here: (1) higher mass resolution on a triple quadrupole mass spectrometer and (2) atmospheric pressure

Table 13.3 Additional rules for discovery (nonGLP) PK assays requiring QC samples (level III). Adapted from [6], with permission from Taylor and Francis Group.

1 Use all the rules for ''full PK - level II'' assays (except rule 7) plus the following rules.

2 Quality control (QC) standards are required and a minimum of six QCs at three concentrations (low, middle, high) are to be used. The QC standards should be frozen at the same freezer temperature as the samples to be assayed.

3 The QC standards need to be traceable to a separate analyte weighing from the one used for the standard curve standards.

4 The standard curve standards should be prepared on the same day the samples are prepared for assay - the standard curve solutions needed for this purpose may be stored in a refrigerator until needed for up to six months.

5 At least two-thirds of the QC samples must be within 25% of their prepared (nominal) values.

6 If dilution of one or more samples is required for this assay, then an additional QC at the higher level must be prepared, diluted and assayed along with the sample(s) needing dilution - this QC should be run in duplicate and at least one of the two assay results must meet the 25% criterion.

photoionization (APPI). The higher mass resolution on a triple quadrupole mass spectrometer has been described in detail recently [88, 139, 140]. This new instrumentation provides an additional mass filtering capability that is not available on conventional triple quadrupole MS systems. It has been demonstrated that this enhanced mass resolution can be very helpful for discovery PK assays when one is approaching the limit of quantitation [88, 139, 140]. The APPI is a new ionization system that uses ultraviolet radiation as part of the ionization step [141151]. The APPI source has the potential to provide improved sensitivity for those compounds that do not ionize well with either APCI or ESI. In a recent study by Cai et al. [143], the authors stated that APPI worked well for many nonpolar compounds, including those that did not ionize well with either APCI or ESI sources.

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