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To develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determining the concentration of posaconazole in dried blood spot (DBS) samples, providing a feasible reference for the application of the dried blood spot method and the clinical practice of posaconazole therapeutic drug monitoring (TDM).
DBS samples were obtained by dropping whole blood samples containing posaconazole onto Whatman 903® cards and drying them. The sample processing procedure involved cutting out a round hole with a diameter of 3 mm to obtain the analytical sample. The sample was extracted by adding 0.2% formic acid-50% acetonitrile (containing an internal standard of 0.50 μg·ml-1), followed by sonication and centrifugation. The supernatant was directly injected for detection. LC-MS/MS was used for determination. The Phonomenex Kinetex XB-C18 column was selected as the chromatographic column, and gradient elution was used for separation. The mass spectrometry detection mode was positive ion multiple reaction monitoring. The monitored ion pairs for the analyte posaconazole and the internal standard D5-posaconazole were 701.3→614.4 and 705.3→618.4, respectively. Full method validation was conducted in accordance with ICH Guideline M10 (Bioanalytical Method Validation and Study Sample Analysis), covering selectivity, specificity, linearity, carryover, accuracy, precision, extraction recovery, matrix effect, analyte stability, together with DBS-specific validation parameters including hematocrit (Hct) impact, spotting volume effect and spot homogeneity. In addition, the developed DBS method was applied to the detection of clinical TDM samples of posaconazole and compared with the plasma method, and the consistency of the two methods was evaluated.
The LC-MS/MS method for the determination of posaconazole in DBS had good selectivity and linearity (0.10-10.00 μg·ml-1), without the influence of cross-talk and carry-over. The recovery rate of the analyte during the sample processing was in the range of 70.67%-75.66%. The intra-batch accuracy and precision were in the ranges of -12.18%--0.86% and 3.05%-8.75%, respectively, and the inter-batch accuracy and precision were in the ranges of -4.21%-3.49% and 8.57%-12.21%, respectively. For the matrix effect tested with six blank whole blood samples from different sources, the accuracy was within ±15% and the precision was all <15%. The stability results showed that posaconazole was stable for at least 3, 3, 14, 30, and 90 days at room temperature (20-25 ℃), 37 ℃, 4 ℃ -20 ℃, and -70 ℃ in DBS samples, respectively. The results of Bland-Altman and Passing-Bablok analyses of 78 clinically verified samples showed that there was a good correlation between the determination results of posaconazole in dried blood spots (DBS) and plasma. However, there was a significant deviation (P<0.001). The determination results in DBS were lower than those in plasma (average difference=0.33 μg·ml-1), and this was more significant in cases with low hematocrit (Hct).
A straightforward and practical LC-MS/MS method was successfully developed and fully validated for posaconazole quantitation in DBS samples. This method requires a minimal blood volume (minimum 10 μL whole blood), features a short analytical runtime (2.00 min per injection) and facilitates streamlined laboratory operation with robust practicability, which can serve as a convenient alternative analytical strategy for routine clinical TDM of posaconazole.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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