Direct Quantitative Bioanalysis of Drugs in Dried Blood Spot Samples

Nov 17, 2009 - Road, Ware, Hertfordshire, SG12 0DP, U.K.. The CAMAG ... of drugs from dried blood spot (DBS) samples, using an MS detector, with or...
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Anal. Chem. 2009, 81, 10275–10284

Direct Quantitative Bioanalysis of Drugs in Dried Blood Spot Samples Using a Thin-Layer Chromatography Mass Spectrometer Interface Paul Abu-Rabie* and Neil Spooner PreClinical Development Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Research and Development, Park Road, Ware, Hertfordshire, SG12 0DP, U.K. The CAMAG thin-layer chromatography mass spectrometer (TLC-MS) interface has been assessed as a tool for the direct quantitative bioanalysis of drugs from dried blood spot (DBS) samples, using an MS detector, with or without high-performance liquid chromatography (HPLC) separation. The approach gave acceptable sensitivity, linearity, accuracy, and precision data for bioanalytical validations with and without the inclusion of HPLC separation. In addition, the direct elution technique was shown to increase assay sensitivity for a range of analytes representing a wide “chemical space” for pharmaceuticaltype molecules over that obtained by conventional manual extraction of samples (punching of DBS and elution with solvent prior to HPLC-MS analysis). Investigations were performed to optimize extraction time, minimize sampleto-sample carry-over, and compare chromatographic performance. On the basis of this preliminary assessment, it has been demonstrated that the TLC-MS interface has the potential to be an effective tool for the direct analysis of drugs in DBS samples at physiologically relevant concentrations, an approach that could provide significant time and cost savings and greatly simplify bioanalytical procedures compared to current manual practices. Further, the increased sensitivity compared to that of manual extraction may enable the analysis of analytes not currently amenable to DBS sampling due to limitations in assay sensitivity. Measuring pharmaceutical concentrations from whole blood is recognized to be a suitable alternative to more traditional plasma analysis by regulatory authorities.1-3 Further, there are numerous reports in the literature of the use of dried blood spots (DBS) for the collection of samples for the quantitative determination of circulating exposures of pharmaceuticals in animal toxicokinetic (TK)4 and clinical pharmacokinetic (PK) and therapeutic drug * To whom correspondence should be addressed. E-mail: Paul.2.Abu-Rabie@ gsk.com. Phone: +44(0) 1920 883995. Fax: +44(0) 1920 884374. (1) ICH Harmonised Tripartate Guideline, Code S3A Toxicokinetics: the assessment of systemic exposure in toxicity studies, 1995. (2) ICH Common Technical Document Code M4. The common technical document for the registration of pharmaceutical for human use, 1999. (3) FDA. Fed. Regist. 1995, 60, 11264-11268. (4) Barfield, M.; Spooner, N.; Lad, R.; Parry, S.; Fowles, S. J. Chromatogr., B 2008, 870, 32–37. 10.1021/ac901985e  2009 American Chemical Society Published on Web 11/17/2009

monitoring studies at physiologically relevant concentrations.5-7 The surge in interest in DBS techniques for supporting pharmaceutical exposure studies is due to the many advantages it offers over conventional plasma sampling. These include the reduction in blood volumes required, with associated cost and ethical advantages, the simplification of clinical sampling procedures, and the reductions in sample processing, storage, and transportation costs.4-7 However, it is notable that these benefits are not necessarily transferred into the bioanalytical laboratory. For example, the requirement to punch a disk out of the DBS sample and extract with solvent for several minutes prior to highperformance liquid chromatographic (HPLC) separation, followed by selected reaction monitoring (SRM) tandem mass spectrometric (MS/MS) detection is laborious and time-consuming, compared to the approaches that are routinely used in the quantitative analysis of plasma samples, i.e., automated pipetting followed by sample preparation using protein precipitation with organic solvent, solid-phase extraction, liquid-liquid extraction, etc. A suitable direct analysis technique for DBS samples could readily counter these disadvantages, potentially resulting in a process that is simpler than those currently used for plasma analysis (Figure 1). Further, significant additional cost and time savings and further gains in simplicity could be achieved if the direct analysis technique also eliminated the need for liquid chromatographic separation of the sample. A number of surface sampling techniques exist, that in conjunction with MS detection could potentially be used for the direct quantitative analysis of DBS samples.8 Applicability of highthroughput direct analysis techniques with DBS has been claimed, but routine application of these techniques has yet to be reported. Direct analysis in real time (DART) has reportedly been used to detect endogenous and exogenous substances in whole blood on glass slides.9 However, it is thought that blood is not well-suited for this type of analysis without prior sample preparation,10 and (5) Spooner, N.; Lad, R.; Barfield, M. Anal. Chem. 2009, 81, 1557–1563. (6) Ramakrishnan Y.; Dewit O.; Miller S.; Barfield M.; Spooner N. Br. J. Pharmacol., submitted for publication, 2009. (7) Edelbroek, P. M.; Van der Heijden, J.; Stolk, L. M. L. Ther. Drug Monit. 2009, 31, 327–336. (8) Van Berkel, G. J.; Pasilis, S. P.; Ovchinnikova, O. J. Mass Spectrom. 2008, 43, 1161–1180. (9) Cody, R. B.; Laramee, J. A.; Durst, H. D. Anal. Chem. 2005, 77, 2297– 2302. (10) JEOL Application Note: AccuTOF with DART, Analysis of Biological Fluids. http://www.jeolusa.com/RESOURCES/AnalyticalInstruments/DocumentsDownloads/tabid/337/Default.aspx?EntryId)44 (accessed Aug 12, 2009).

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Figure 1. Schematic showing the differences between the different modes used for DBS samples analysis during the evaluation of the TLC-MS interface.

our own investigations have yet to show any promise using this technique with DBS (unpublished data). Desorption electrospray ionization (DESI) has been reported as being a promising method for the analysis of dried blood,11 and direct analysis of DBS on paper using DESI for the screening of neonatal metabolic disorders has been reported.12 However, in our experience both DART and DESI appear at least an order of magnitude away from the sensitivity required for existing assays of drugs at physiologically relevant concentrations. An alternative approach is the direct online solvent desorption of analytes from DBS samples. Deglon et al. successfully used online solvent elution of a punched DBS followed by HPLC-MS detection for the quantitative analyses of a number of drug molecules.13 However, this approach required the prepunching of DBS samples and the mounting of the punched sample in an online holder. An alternative approach is the use of the CAMAG thin-layer chromatography (TLC)-MS interface. This instrument was designed to extract compounds from a TLC plate and feed them directly into a mass spectrometer for substance identification, structure elucidation, and quantification.14-16 Van Berkel and Kertesz have demonstrated the potential use of this interface for (11) Takat, Z.; Wiseman, J.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471– 473. (12) Takats, Z.; Wiseman, J.; Cooks, R. G. J. Mass Spectrom. 2005, 40, 1261– 1275. (13) Deglon, J.; Thomas, A.; Cataldo, A.; Mangin, P.; Staub, C. J. Pharm. Biomed. Anal. 2009, 49, 1034–1039. (14) Luftmann, H. Anal. Bioanal. Chem. 2004, 378, 964–968. (15) Aranda, M.; Morlock, G. Rapid Commun. Mass Spectrom. 2007, 21, 1297– 1303. (16) Luftman, H.; Aranda, M.; Morlock, G. E. Rapid Commun. Mass Spectrom. 2007, 21, 3772–3776.

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the direct elution and MS detection of DBS samples and thin tissue sections.17 This manuscript describes further preliminary investigations of the TLC-MS interface for the direct quantitative analysis of drugs in DBS samples with and without a HPLC column for a range of test compounds, representing a wide “chemical space” for pharmaceutical-type molecules (Supporting Information Figure S1). Data is presented on the optimization of sample extraction time, and the chromatographic performance is compared to that obtained by routine manual extraction of DBS samples, involving punching out a disk, followed by solvent extraction and HPLC-MS/ MS analysis. Quantitative performance was assessed by the determination of sensitivity, linearity, accuracy, and precision. Further, the use of an improved wash step to decrease the carryover and, hence, improve the quantitative assay performance at lower analyte concentrations was investigated. For descriptive purposes the term “direct extraction” will be used in this paper when the TLC-MS is used with a HPLC column and “direct analysis” will be used where the column is excluded and an “MS response” rather than a chromatographic peak is generated (Figure 1). EXPERIMENTAL SECTION Chemical Reagents and Equipment. Methanol, acetonitrile, and water were of HPLC gradient grade and were obtained from Fisher Scientific Ltd. (Loughborough, U.K.). All other chemicals were of AnalaR grade, supplied by VWR International Limited (Poole, U.K.). Control cynomologus monkey blood was obtained from Harlan (Hull, U.K.). Control human blood was supplied by GlaxoSmithKline (GSK) volunteers in accordance with current GSK polices on informed consent and ethical approval. (17) Van Berkel, G. J.; Kertesz, V. Anal. Chem. 2009, 81, 9146–9152.

Figure 2. Schematic diagram of direct extraction/analysis assembly and operation of the TLC-MS interface.

Paracetamol (acetaminophen), SB243213, [2H4]-acetaminophen, sitamaquine, and [2H10]-sitamaquine were obtained from GSK (Stevenage, U.K.). Ibuprofen and 4-nitrophthalic acid were obtained from Sigma-Aldrich (Poole, U.K.). Proguanil was obtained from Molekula (Dorset, U.K.). Simvastatin was obtained from Calbiochem (Nottingham, U.K.). Benzethonium chloride was obtained from Alfa Aesar (Lancs, U.K.). Ahlstrom grade 237 paper for blood spots was supplied by ID Biological Systems (Abbots Langley, U.K.). Sample tubes were obtained from Micronics (Sanford, U.S.A.). The centrifuge (model 5810R) was supplied by Eppendorf (Hamburg, Germany). Harris punch and cutting mat were supplied by Ted Pella (Redding, U.S.A.). Benchtop sample shaker (model HS 501 D) was supplied by Janke and Kunkel, IKA Labortechnik (Staufen, Germany). The CAMAG TLC-MS interface was obtained from Omicron Research Ltd. (Wiltshire, U.K.). The HPLC-MS/MS system consisted of an Agilent 1100 binary pump (Palo Alto, CA) with integrated column oven. MS detection was by a Sciex API-3000 (Applied Biosystems/MDS Sciex, Canada) equipped with Turbo IonSpray source. HPLC-MS/MS and MS/MS data were acquired and processed (integrated) using Analyst software (v1.4.2 Applied Biosystems/MDS Sciex, Canada). Preparation of Test Samples. Three bioanalytical DBS test methods were used for assessing the performance of the TLC-MS interface; two single-analyte methods were used for paracetamol and sitamaquine together with a multianalyte cassette method for a suite of compounds that was designed to represent a wide chemical space (ibuprofen, 4-nitrophthalic acid, paracetamol (all analyzed in negative ion mode) and simvastatin, sitamaquine, benzethonium chloride, and proguanil (positive ion mode)). The single-analyte assays used stable isotopically labeled analogues as internal standards (IS; [2H10]-sitamaquine and [2H4]-paracetamol), whereas the multianalyte assay used SB243213 as an IS (monitored separately in positive and negative ion modes). Primary stock solutions for each test compound and IS were prepared in dimethylformamide (DMF, 10 mg/mL for paracetamol, 1 mg/mL for all other compounds). For each assay, working standards at suitable concentrations were made up in methanol/ water (1:1, v/v).

For the manual extraction experiments, analytical samples were prepared by diluting the appropriate working solutions with blank control blood, whereas for the direct extraction and analysis experiments samples were prepared by diluting the appropriate working solutions with control blood containing a suitable concentration of IS. In all cases, the spiking volume into blood was less than 5% nonmatrix solvent. For the single-analyte assays (sitamaquine and paracetamol), calibration standards and quality control (QC) samples were prepared over a concentration range relevant for the physiological exposure of these drugs. For sitamaquine, the concentrations of calibrants were 5, 10, 20, 50, 100, 200, 500, 800, and 1000 ng/mL and QCs were 5, 20, 100, 800, and 1000 ng/mL. For paracetamol, the concentrations of calibrants were 50, 100, 200, 500, 2000, 5000, 10 000, 20 000, 40 000, and 50 000 ng/mL and QCs were 50, 200, 2500, 40 000, and 50 000 ng/mL. For the multianalyte method, all analytes were prepared at 4000 ng/mL only. Comparative sensitivity only was tested for these compounds. DBS samples were prepared by spotting a fixed volume (15 µL) of blood onto the paper and drying for at least 2 h at room temperature. If required, samples were stored at room temperature in a sealed plastic bag containing desiccant. Manual Extraction Procedure. A 3 mm diameter disk was punched from the center of the DBS sample into a clean tube. This was then extracted by the addition of 100 µL methanol containing the appropriate IS. The tube was shaken using a benchtop shaker for 1 h, centrifuged for 10 min at 3000g to push the disk to the bottom of the tube, and the supernatant was then transferred to a clean sample tube. An aliquot (2-5 µL) of this extract was then injected onto the HPLC-MS/MS system. Direct Extraction and Direct Analysis Using the TLCMS Interface. For direct extraction experiments an HPLC column was present in the system. For direct analysis, the HPLC column was absent. In both cases, the TLC-MS interface (Figure 2) was located between the HPLC pump delivering solvent and a HPLC column (when present) coupled to the MS system. The DBS sample was placed on the horizontal platform directly under the TLC-MS interface extraction head, which was lowered onto the Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

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sample to create a 4 mm diameter seal on the center of the spot. A manual bypass/extraction switch controls solvent flow. In bypass position the solvent flows from the HPLC pump directly to the HPLC column (when present) and MS system. In extract position, the solvent flows down the inlet of the TLC-MS interface extraction head onto the surface of the spot, then up the outlet and on to the HPLC column (when used) and MS system. Direct extraction/analysis was performed by simultaneously starting MS data acquisition, the HPLC solvent gradient (where used), and switching the TLC-MS bypass/extraction valve to the “extract” position, thus diverting the solvent flow onto the blood spot surface. A variety of extraction times (the length of time solvent was allowed to flow across the DBS sample surface) were investigated. HPLC-MS/MS Analysis. For manual extraction and direct extraction experiments, the following HPLC conditions were used: The sitamaquine single-analyte assay used a 50 mm × 2.0 mm i.d. Varian Polaris C18 5 µm HPLC column (Palo Alto, CA), a flow rate of 500 µL/min, column temperature of 40 °C, run time of 1.5 min, and isocratic chromatography; 62:38 (v/v) 10 mM methyl ammonium acetate (pH 4.2/acetonitrile. The paracetamol single-analyte assay used a 50 mm × 4.0 mm i.d. YMC-Pack ODS-AQ 3 µm HPLC column (Dinslaken, Germany), a flow rate of 800 µL/min, column temperature of 40 °C, run time of 2.5 min, and gradient chromatography employing the mobile phases ammonium acetate (1 mM, native pH) (A) and methanol (B). Following sample injection, the mobile phase was held at 100% A for 0.08 min. A ballistic gradient to 0% A at 1.08 min was followed by an isocratic period at 0% A to 1.25 min. The mobile phase was then returned to 100% A by 1.26 min and was held as this composition until 2.5 min, before the injection of the next sample. The multianalyte assay samples were run twice, once in positive ion MS mode and then in negative ion mode. Positive ion mode used a 50 mm × 2.1 mm i.d. Hypurity C18 3 µm HPLC column (Thermo Scientific, Massachusetts), a flow rate of 1000 µL/min, column temperature of 40 °C, run time of 3.8 min, and gradient chromatography employing the mobile phases ammonium acetate (1 mM, native pH) (A) and acetonitrile (B). Following sample injection, the mobile phase was held at 95% A for 0.1 min. A ballistic gradient to 20% A at 1.00 min was followed by an isocratic period at 20% A to 3 min. The mobile phase was then returned to 95% A by 3.2 min and was held as this composition until 3.8 min, before the injection of the next sample. Negative ion mode used a 50 mm × 4.6 mm i.d. Hypurity C18 3 µm HPLC column (Thermo Scientific, Massachusetts) a flow rate of 1000 µL/min, column temperature of 40 °C, run time of 3.0 min, and gradient chromatography employing the mobile phases ammonium acetate (1 mM, native pH) (A) and acetonitrile (B). Following sample injection the mobile phase was held at 95% A for 0.1 min. A ballistic gradient to 10% A at 1.0 min was followed by an isocratic period at 10% A to 2 min. The mobile phase was then returned to 95% A by 2.1 min and was held as this composition until 3.0 min, before the injection of the next sample. For all methods, the HPLC eluent was introduced into the MS interface using a 1-in-4 split ratio. MS data was acquired in SRM mode. The transitions monitored are detailed in Supporting Information Table S1. MS source conditions were optimized to 10278

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give the maximum response for a given analyte/assay. For the single-analyte sitamaquine and paracetamol analyses, concentrations of test compounds were determined from the peak area ratios of analyte to IS using Analyst software. For the multianalyte assays, a single-concentration sample was analyzed and analyte-to-IS peak area ratio response was compared between the different sample preparation techniques. RESULTS AND DISCUSSION Direct ExtractionsOptimization of Extraction Time. The effect of TLC-MS interface extraction time during direct extraction chromatography (with HPLC column present) was tested by running 100 ng/mL sitamaquine DBS samples with extraction times ranging from 0.5 to 60 s. Analyte peak height does not increase significantly beyond ∼8 s (Figure 3A). Similarly, analyte peak area increases up to ∼10 s, after which the response plateaus (Figure 3B). Peak area ratio gives a relatively flat response with increasing extraction time, indicating the IS is performing well (Figure 3C). Retention time shows no significant change with increasing extraction time (Figure 3D). Chromatographic peak shape was acceptable up to 5 s extraction, after which it became increasingly broad (Supporting Information Figure S2). The number of theoretical plates decreases steadily up to an extraction time of ∼10 s, after which the value plateaus (Figure 3E). The number of theoretical plates, N, was calculated using the following equation where tR is the retention time and W1/2 is the peak width at half-height.

( )

N ) 5.55

tR W1/2

2

Analyte peak asymmetry (a measure of peak tailing) is shown to increase with increasing extraction time (Figure 3F). Analyte peak asymmetry was calculated as follows: peak asymmetry ) (peak end time) (retention time)/(retention time) - (peak start time) On the basis of the outcome of these tests, an extraction time of 2 s was used for most subsequent direct extraction experiments, as it gave a good compromise between chromatographic performance, analyte response, and the robustness of the system. It should be noted that some of the variation observed in the above responses can be attributed to the manual nature of the extraction process and the associated human timing error. Human timing error may have occurred at both the start of extraction (based upon the start of the data acquisition) and the duration of extraction. Direct ExtractionsAssay Sensitivity. The relative HPLC-MS/ MS sensitivity for the analysis of DBS samples by direct extraction chromatography using the TLC-MS interface was determined by comparing the response with that of manually extracted samples. Identical HPLC and MS conditions were used for both the manually extracted samples and directly extracted samples using the TLC-MS interface. An extraction time of 2 s was used for the direct extraction analyses. For manual extraction experiments, the injection volume was optimized to give the maximum MS response (at least 5:1 signal-to-noise at the LLQ), while still ensuring a linear response across the calibration range and

Figure 3. Charts showing trends in chromatographic performance with varying extraction times, when using direct extraction chromatography with the TLC-MS interface and HPLC separation. Single-analyte sitamaquine (100 ng/mL) human DBS samples were used for all analyses: (A) effect on sitamaquine HPLC-MS/MS peak height response; (B) effect on sitamaquine HPLC-MS/MS peak area response; (C) effect on sitamaquine to internal standard ([2H10] sitamaquine) peak area ratio response; (D) effect on retention time; (E) effect on number of theoretical plates; (F) effect on sitamaquine peak asymmetry.

producing an acceptable peak shape. For the test compounds used this was either 5 µL (single-analyte sitamaquine) or 2 µL (all other assays). Figures 4-6 compare HPLC-MS/MS performance for manual extraction and direct TLC-MS extraction of a range of test compounds. Two important observations should be noted. First, direct TLC-MS extraction is shown to retain good analyte retention and peak shape compared to that obtained using manual extraction HPLC-MS/MS methods. Second, a large increase in response of between 3.5- and 16-fold for both peak heights and areas was observed for all test compounds using direct extraction compared to manual extraction (Table 1). However, it is notable that this increase was only a small proportion of the theoretical maximum possible increase in response, i.e., between 4% and 45%

(see calculations in the Supporting Information). It is therefore apparent that the initial 2 s extraction introduces only a small portion of the analyte available. To investigate this further, an experiment was performed where the TLC-MS extraction head was left “sealed” on a 100 ng/mL sitamaquine DBS sample and the same sampling area was extracted for 2 s on six successive occasions approximately 1.5 min apart, using the chromatographic conditions for the sitamaquine single-analyte assay (Figure 7 and Table 2). On the basis of the areas under these six peaks, the first 2 s extraction elutes 17.2% of the analyte (assuming the amount of compound remaining after the sixth extraction is negligible) (Table 2). Note this test does not take carry-over into account and is only intended to provide a rough estimate. Analytical Chemistry, Vol. 81, No. 24, December 15, 2009

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Figure 4. Comparison of mass chromatograms obtained by manual and direct extraction analyses of single-analyte paracetamol (left) and sitamaquine (right) human DBS samples. Manual extraction involved the extraction of a 3 mm punch taken from DBS sample with 100 µL methanol and injection of an aliquot onto the HPLC-MS/MS system. Direct extraction involved the extraction of the DBS sample with the TLC-MS interface for 2 s, followed by HPLC-MS/MS analysis.

From the outcome of this experiment, it is clear that the direct extraction TLC-MS technique has the potential to give greater assay sensitivity than manual extraction. For the manual extraction method, 100 µL is the minimum practical volume of methanol that can be used to elute a 3 mm DBS punch. Further, it is unlikely that greater than 20 µL could be injected onto the HPLC column while maintaining good chromatographic performance (2-5 µL is more typical). The manually extracted sample could be dried down and reconstituted, but this is a longer process and risks not being able to get the entire extracted sample back into solution. For the direct extraction chromatography technique, it is possible that optimization of mobile phase, longer extraction times, prewetting of the area to be sampled, and a more efficient extraction head (designed specifically for DBS samples) could further increase assay sensitivity. Direct ExtractionsLinearity, Accuracy, Precision, and Carry-Over. Test bioanalytical validations were run for the singleanalyte sitamaquine and paracetamol methods using direct extraction with the TLC-MS interface. These consisted of two calibration lines at the start and end of the analytical run, bracketing four replicate QC samples at each of five concentrations spanning the calibration range. Calibration plots of analyte/IS peak area ratio versus the nominal concentration of the analyte in blood were constructed, and a weighted 1/x2 linear regression was applied to the data for both analytes. Acceptable linearity was achieved for sitamaquine over the range of 5-1000 ng/mL and paracetamol over the range of 50-50 000 ng/mL. The linear regression equation for sitamaquine was y ) 0.00148x - 0.000158, r2 ) 0.9970. The linear regression equation for paracetamol was y ) 0.0236x + 1.13, r2 ) 0.9950. The QC accuracy and precision data for sitamaquine and paracetamol were generally within internationally recognized acceptance criteria for assay validations18 and within the pre(18) Shah, V. P.; Midha, K. K.; Findlay, J. W. W.; Hill, H. M.; Hulse, J. D.; McGilvery, I. J.; McKay, G.; Miller, K. J.; Patnik, R. N.; Powell, M. L.; Tonicelli, A.; Vishwanathan, C. T.; Yacobi, A. Pharm. Res. 2000, 17, 1551– 1557.

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defined ±15% limits (Supporting Information Tables S2 and S3). The exception was at the LLQ, where initially a very high positive bias was observed for both analytes. This was found to be due to the considerable carry-over contamination that occurred after the analysis of an HLQ standard in the first calibration line. In its current format the TLC-MS interface does not have a built-in wash step to deal with the sort of contamination that occurs when extracting a DBS sample. A suitable wash step procedure was identified, involving manually reconfiguring the system to back flush the TLC-MS interface extraction head. When the sitamaquine test validation was repeated and this wash step was incorporated, data well within the acceptance criteria was obtained at the LLQ (Supporting Information Table S2). Direct Analysis. For the purposes of this manuscript “direct analysis” refers to direct extraction of DBS using the TLC-MS interface with the extract going directly to the MS, i.e., no HPLC column is included. Only the single-analyte sitamaquine samples were assessed using direct analysis. Since no chromatographic separation is taking place for direct analysis, the liquid used to extract the DBS is referred to as the “extraction solvent”, rather than mobile phase. Similarly the assay is generating a “MS response” rather than a chromatographic peak. For the purposes of this investigation the area under the response was integrated in the same way a chromatographic peak would be to generate sensitivity, accuracy, and precision data. Two extraction solvents were used to assess the direct analysis of sitamaquine. The first was the isocratic mobile phase (62:38 (v/v) 10 mM methyl ammonium acetate (pH 4.2)/acetonitrile) which was also used in the manual extraction of sitamaquine. Although this produced excellent linearity, accuracy, and precision data, there was not enough sensitivity to produce adequate signalto-noise for the lower concentration samples on the MS used (data not shown). A usable method using this extraction solvent may be possible on a more sensitive instrument. Switching the extraction solvent to 70:30 (v/v) methanol/water gave a considerable gain in sensitivity and produced adequate signal-to-noise for the lower concentration samples (Supporting Information Figure S3). Studies in our laboratories have shown

Figure 5. Comparison of mass chromatograms obtained by manual and direct extraction analyses of multianalyte cynomologus monkey DBS samples in negative ion mode. Manual extraction involved the extraction of a 3 mm punch taken from DBS sample with 100 µL methanol and injection of a 2 µL aliquot onto the HPLC-MS/MS system. Direct extraction involved the extraction of the DBS sample with the TLC-MS interface for 2 s, followed by HPLC-MS/MS analysis.

that 70:30 methanol/water (v/v) is particularly effective at eluting compounds from DBS samples by manual extraction. It is notable that the response (peak height) for direct analysis using the TLC-MS interface without a column present was similar to that obtained using direct extraction with a column present (Supporting Information Figure S4). Direct AnalysissLinearity, Accuracy, and Precision. Test validations were run using 2 and 20 s extraction times for sitamaquine. The extraction solvent was 70:30 (v/v) methanol/ water, and the back-flush wash step was incorporated. Calibration plots of analyte/IS peak area ratio versus the nominal concentration of sitamaquine in blood were constructed, and a weighted 1/x2 linear regression was applied to the data for both analytes. Both calibration lines showed that the response over the assay range of 5-1000 ng/mL was linear. The linear regression equation for 2 s extraction was y ) 0.00138x - 0.000137, r2 )

0.9964. The linear regression equation for 20 s extraction was y ) 0.00164x + 0.00124, r2 ) 0.9973. DBS QC samples at concentrations of 20 and 800 ng/mL were analyzed. Accuracy and precision data (Supporting Information Table S4) generated from the 20 s extraction run was better than that of the 2 s extraction, with impressively good accuracy (