Estimation and Correction of the Blood Volume Variations of Dried

May 24, 2016 - ... of Genomic Medicine, National Taiwan University, Taipei 10051, Taiwan. § ... ABSTRACT: Dried blood spots (DBSs) have had a long hi...
0 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Estimation and Correction of the Blood Volume Variations of Dried Blood Spots Using a Postcolumn Infused-Internal Standard Strategy with LC-Electrospray Ionization-MS Hsiao-Wei Liao,†,‡ Shu-Wen Lin,§,⊥ Guan-Yuan Chen,†,‡ and Ching-Hua Kuo*,†,‡,⊥ †

School of Pharmacy, College of Medicine, National Taiwan University, Linsen S. Rd., Chongsheng, Taipei 10051, Taiwan The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taipei 10051, Taiwan § Graduate Institute of Clinical Pharmacy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan ⊥ Department of Pharmacy, National Taiwan University Hospital, Taipei 10051, Taiwan ‡

S Supporting Information *

ABSTRACT: Dried blood spots (DBSs) have had a long history in disease screening in newborns but have gained attention in recent years in the medical care of adults because of the growing importance of personalized medicine. DBSs have several advantages, such as easy transportation, cost-effectiveness, and minimally invasive biological sampling. There are two strategies to process DBS samples. One method takes a fixed diameter of subsample, and another requires the extraction of the whole spot. The whole-spot extraction method is less affected by hematocritcaused errors, but it requires calibration of the blood volume. We propose a novel strategy using a postcolumn infused-internal standard (PCI-IS) method with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for estimating and correcting blood volume variations on the DBS cards. By using PCI-IS to measure the extent of ion suppression in the first ion suppression zone in the chromatogram, the blood volume on the DBS cards can be calculated and further calibrated. We used reference blood samples with different volumes (5 to 25 μL) to construct a calibration curve between the blood volume and the extent of ion suppression. The calibration curve was used to estimate the blood volume on the DBS cards collected from 6 volunteers, with 5 designated volumes from each volunteer. The estimation accuracy of the PCI-IS method was between 74.5% and 120.3%. The validated PCI-IS method was used to estimate and calibrate blood volume variation and also to quantify the voriconazole concentration for 26 patients undergoing voriconazole therapy. A high correlation was found for the quantification results between the DBS samples and the conventionally used plasma samples (r = 0.97). The PCI-IS method was demonstrated to be a simple and accurate method for estimating and calibrating the blood volume variation on DBS cards, which greatly facilitates using the DBS method for therapeutic drug monitoring (TDM) for improving the efficacy and safety of drug therapy.

T

to the conventional venous blood sampling approach, the DBS method provides a more convenient and cost-effective way for biological sampling.6−8 The growing importance of personal medicine makes the DBS sampling approach a method of high interest for clinical therapeutic drug monitoring (TDM) because patients can sample their blood at home or at local hospitals in order to control the efficiency and safety of a treatment.1,3,9,10 The advantages of ease and the stable transportation of such samples also resolve problems for hospitals that do not have enough equipment for the preparation and storage of plasma samples.

he dried blood spot (DBS) sampling technique has a long history that can be traced back to 1960. The initial application of this technique was mainly for screening for diseases, such as homocystinuria, maple syrup urine disease, and congenital adrenal hyperplasia, in newborns because of its minimally invasive procedure of a finger prick or heel.1−3 A simple spot of a small amount of blood is collected on a filter paper card and then treated with a drying process, allowing the dried blood samples to be easily transported to laboratories for further analysis.3 In general, the dried blood sample can remain stable under suitable storage conditions or under refrigeration for months to years, and this method can increase the stability of photosensitive compounds.4,5 The additional advantages of the DBS technique include low biohazard risk during the shipment of samples and a smaller sampling volume. Compared © XXXX American Chemical Society

Received: March 22, 2016 Accepted: May 23, 2016

A

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Moreover, the PCI-IS method can simultaneously be used to correct the matrix effect (ME) caused quantification errors of the target analytes for the DBS sample. To evaluate the applicability of the PCI-IS method for correcting blood volume variations and ME-caused quantification errors, the developed method was applied to voriconazole TDM samples. Voriconazole was the first available second-generation triazole drug for the treatment of serious invasive fungal infections (IFIs).23 Genetic polymorphisms, age, liver disease, and comedications may cause interindividual variability of the voriconazole pharmacokinetics,24,25 and TDM is highly recommended to improve the efficacy and safety of the therapy in clinical practice. The quantitative results obtained by the DBS sampling technique were compared with the conventionally used venous blood sampling technique to demonstrate the applicability of the PCI-IS method for TDM sample analysis.

There are two strategies for processing DBS samples. One takes a fixed diameter subsample, and another requires wholespot extraction.9,11,12 The fixed diameter approach uses a punch to ensure the same blood volume is being taken from the DBS for concentration analysis. However, the varying hematocrit (HCT) of the test individuals will significantly alter analytical uncertainty.13 The individual difference in the HCT values affects the viscosity (fluidity) of blood, which results in differences in the extent of blood spreading on the DBS card. This is one of the main sources of HCT-based bias. Abu-Rabie et al. have proposed the use of an isotope internal standard spraying technique to calibrate HCT-based bias.9,14 Instead of using a fixed diameter subsample of the DBS sample for concentration analysis, the whole-spot extraction approach takes the whole blood spot for analysis. The whole-spot approach is less affected by the HCT-based bias. However, how to control for the blood volume being analyzed becomes the bottleneck of this approach. Some laboratories use pipets to accurately control the volume of the blood samples on the DBS card, but this approach conflicts with the advantages of the DBS method in terms of self-sampling, simplification, and economics. Some techniques, such as the DBS system Mitra or volumetric absorptive microsampling, have been introduced to control the blood volume on DBS cards, but another device is required for DBS volume control. Therefore, there is a great demand for an effective method to control or estimate the blood volume on DBS cards. Due to the small sampling volume (approximately 15 ± 5 μL per spot), a lower assay sensitivity is one of the main limitations of the DBS technique.9 The high sensitivity and selectivity of liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) makes the technique a perfect match for coupling with DBS analysis. Many studies have proven LC-ESIMS combined with the DBS technique is an effective and efficient platform for the quantitation of pharmaceuticals for TDM and pharmacokinetics studies.7,15 The postcolumn introduction of an internal standard in LC-ESI-MS was originally proposed by Choi et al.16 and Cheng and Tsai17 to correct the quantitative errors associated with matrix signal suppression. We previously demonstrated that the postcolumn infused-internal standard (PCI-IS) is an economical and effective method for improving the quantification accuracy of analytes in biological samples when using LC-ESI-MS.18−20 Gonzalez et al. recently applied this strategy in small-molecule profiling for an LC-TOF platform. By selecting the appropriate PCI-ISs, they observed a dramatic improvement in the results for the matrix effect.21 In this study, we used the PCI-IS method with LC-ESI-MS to estimate and correct the blood volume variation on DBS cards. Blood samples are isotonic solutions,22 and the total salt content in blood can be used to estimate blood volume. The osmolarity of plasma mostly comes from the contribution of sodium and its counteranions.22 Because of the nonretention ability and strong ion suppression characteristics of blood salts, we can use the PCI-IS method to measure the signal of the first significant ion suppression zone in the LC-ESI-MS chromatogram to estimate the total salts. By using reference blood samples to construct a nonlinear calibration curve between the extent of ion suppression of PCI-IS and the blood volume on DBS cards, the unknown blood volume on the DBS cards can be estimated. The estimated blood volume using the PCI-IS method can be further used to calibrate drug concentrations in the blood using the whole-spot DBS extraction strategy.

■

EXPERIMENTAL SECTION Chemicals and Materials. Voriconazole, posaconazole, sulfamethoxazole, and formic acid (99%) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Ephedrine, flunitrazepam, and butorphanol were purchased from Cerilliant Corporation (Round Rock, TX). Hexakis (1H,1H,3H-perfluoropropoxy) phosphazene (HKP) was purchased from Apollo Chemical Co. (Graham, NC). Acetonitrile (ACN) was obtained from J.T. Baker (Phillipsburg, NJ). Mass-grade methanol was purchased from Scharlau Chemie (Sentmenat, Barcelona, Spain). Whatman 903 Protein Saver cards (Maidstone, UK) were used for the collection of blood samples (DBS samples). The puncher was purchased from a local store, and the size is 3 mm. UHPLC-ESI-MS System. The LC separations were performed using an Agilent 1290 UHPLC system equipped with a binary solvent pump, an autosampler, a sample reservoir, and a column oven, and an Agilent 1260 quaternary solvent pump was used for the postcolumn infusion of the PCI-IS. The mass spectrometer was an Agilent 6460 triple quadrupole system (Agilent Technologies, Waldbronn, Germany). The PCI-IS (posaconazole) was dissolved in ACN at 100 ng mL−1, and the PCI-IS was introduced into the ESI interface at a 0.1 mL min−1 flow rate. A Kinetex C18 2.1 × 50 mm (2.6 μm) column (Phenomenex, Torrance, USA) was employed for the separations. The mobile phase A was 0.1% aqueous formic acid in deionized water. The mobile phase B consisted of 0.1% formic acid in ACN. A 0.3 mL min−1 linear gradient elution was used: 0−1.0 min, 2% mobile phase B; 1.0−3.0 min, 20−95% mobile phase B; 3.0−4.0 min, 95% mobile phase B. The column re-equilibration used 100% mobile phase B for 1.2 min. The sample reservoir and the column oven were maintained at 4 and 40 °C, respectively. The injection volume was 5 μL. The positive electrospray ionization mode was utilized with the following parameters: 325 °C dry gas temperature, 7 L min−1 dry gas flow rate, 45 psi nebulizer pressure, 325 °C sheath gas temperature, 11 L min−1 sheath gas flow rate, 3500 V capillary voltage, and 500 V nozzle voltage. The MS acquisition was executed using the multiple reaction monitoring (MRM) mode. In positive mode, the transitions for voriconazole, posaconazole (PCI-IS), ephedrine (PCI-IS), flunitrazepam (PCI-IS), butorphanol (PCI-IS), and HKP (PCI-IS) were m/z 350.0 → 224.1, 701.3 → 683.3, 166.1 → 148.1, 314.1 → 268.0, 328.2 → 310.2, and 922.0 → 790.1, respectively. In negative mode, the transitions for sulfamethoxazole (PCI-IS) and HKP (PCI-IS) were m/z 252.0 → 155.9 and 966.0 → 806.0, respectively. The B

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry concentrations of posaconazole, ephedrine, flunitrazepam, butorphanol, sulfamethoxazole, and HKP used as the PCI-IS were 100, 100, 100, 100, 50, and 200 ng mL−1, respectively. The PCI-IS flow rate was 0.1 mL min−1 for all the PCI-IS samples. DBS and Plasma Sample Preparation Procedures. The voriconazole and posaconazole standards were prepared separately in methanol at a concentration of 500 μg mL−1 as the stock solution. The working solution was prepared by spiking an appropriate amount of each analyte stock solution into 100% methanol to obtain 100 μg mL−1 of the diluted working solution. The blood samples from healthy volunteers were obtained through peripheral veins via syringe collection and collected in EDTA tubes to prevent coagulation. To prepare voriconazole spiked blood samples, 5 μL of voriconazole working solution was spiked into 50 μL of blood sample. Twenty microliters of the spiked blood sample was spotted onto a DBS card within 1 h after preparation of the spiked blood samples. The spotted cards were air-dried for 2 h. The blood spot on the DBS cards was cut into a clean Eppendorf tube using a 3 mm manual puncher several times until the whole blood spot was cut. Then, 200 μL of methanol was added to extract the cut blood spot, and the sample was extracted using a Geno/Grinder at 1000 rpm for 5 min without beads. After centrifugation at 15 000 rcf for 5 min, the supernatant was collected, filtered with the regenerated cellulose membrane (RC-4, Sartorius, Göttingen, Germany), and stored at −20 °C before UHPLC-ESI-MS analysis. To prepare blood models with different HCTs, the collected blood samples were centrifuged at 3000 rcf for 5 min to obtain an upper plasma layer and lower blood cell layer. Through mixing of adequate volumes of blood cell with plasma, we can create blood models with different HCTs. In this study, we generated 25%, 50%, and 75% HCT blood models as examples for testing the HCT influence on the DBS extraction recovery. To process the plasma samples, protein precipitation was performed by mixing 100 μL of the plasma sample with 400 μL of methanol. The deproteinized sample was centrifuged at 15 000g for 5 min, and the supernatant was then filtered through a 0.22 μm regenerated cellulose membrane before UHPLC-ESI-MS/MS analysis. Using the PCI-IS Method for Blood Volume Estimation on DBS Cards. The PCI-IS method in this study was used to estimate the blood volume of the DBSs. The minimum response of the PCI-IS chromatogram at the first ion suppression zone (Figure 1) was used to represent the degree of ion suppression caused by the salts in the blood samples. Moreover, the individual difference of the MEs can also be corrected by the PCI-IS at each time point. The detailed description of the calibration of the ME-caused quantification errors are provided in our previous publication.19,20 In short, the analyte signal intensities at every time point in the chromatogram were divided by the PCI-IS responses at the same retention times, and the ratios were used to generate the new adjusted chromatogram. The peak area obtained from the new adjusted chromatogram was used for voriconazole quantification. All the MRM chromatograms obtained from the Agilent Triple Quadrupole system were converted into comma separated values (csv) format and processed with Microsoft Excel 2007 (Albuquerque, NM). The information in the csv file

Figure 1. (a) The different blood volumes (5 to 25 μL) on the DBS cards. (b) The overlay of the MRM chromatograms of the PCI-IS (posaconazole) obtained for DBS samples of different blood volumes.

included the mass transition, the retention time, and the signal intensity. The MS acquisition rate was set to 2.46 spectra s−1. Validation of the PCI-IS Method for Quantifying Voriconazole DBS Samples. Aliquots of voriconazole stock solutions were spiked into blank blood samples to obtain 0.3, 0.5, 1, 3, 5, and 10 μg mL−1 voriconazole-spiked blood samples to build a calibration curve used for quantification. The linear curve was created by plotting the corrected ratios of voriconazole with posaconazole (PCI-IS) against the concentration of voriconazole. The limit of detection for each analyte was determined as the concentration at which the signal-tonoise ratio was equal to 3 (S/N = 3). The limit of quantification for each analyte was determined as the concentration at which the signal-to-noise ratio was equal to 10 (S/N = 10). To test the precision and accuracy of the quantification of voriconazole, blood samples obtained from 3 healthy volunteers were spiked with voriconazole at concentrations of 0.3, 1, and 10 μg mL−1. These spiked samples were tested in four runs a day for 3 days. Collection of Clinical Samples. Twenty-six blood samples used for clinical applications were obtained from the National Taiwan University Hospital. This study was approved by the local ethics committee. Signed informed consent was received from all patients who participated in this study. The blood samples were obtained through peripheral venous catheter and collected in EDTA-containing tubes. Five to 15 microliters of the blood samples were spotted on DBS cards, and the rest of the blood sample was centrifuged at 3000 rcf for 15 min to obtain the plasma samples. The plasma samples were stored at −80 °C until use. Quantification of Voriconazole Concentration in Patient Plasma. To quantify the level of voriconazole in the plasma of patients, aliquots of the voriconazole stock solution were spiked into blank plasma samples to obtain 0.3, 0.5, 1, 3, 5, and 10 μg mL−1 voriconazole-spiked plasma samples to build a calibration curve used for quantification.

■

RESULTS Using the PCI-IS Method To Estimate Blood Volume on DBSs. The PCI-IS method infused an internal standard using a postcolumn approach to sense the matrix composition C

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 2. Correlation between the minimum responses at the first ion suppression zone of the PCI-IS chromatograms and the DBS blood volume for 6 tested PCI-ISs. (a) Ephedrine, (b) flunitrazepam, (c) butorphanol, (d) posaconazole, and (e) HKP were tested in the positive mode, and (f) sulfamethoxazole and (g) HKP were tested in the negative mode. Each point in this figure is presented as average ± standard deviation.

salts should correspond to the blood volume. Therefore, the extent of the ion suppression at the first ion suppression zone in the chromatogram can be used to estimate blood volume on the DBS cards. The PCI-IS is used to measure the extent of ion suppression and to translate it into blood volume on DBS cards. To prove this hypothesis, we used different volumes of blood samples on DBS cards to verify the positive correlation between the extent of the ion suppression measured by PCI-IS and the blood volume. In general, the blood volumes on DBS cards varied from 10 to 20 μL. Five blood volumes collected in collection tubes without anticoagulant, ranging from 5 to 25 μL, were spotted on DBS cards through pipetting (Figure 1a). After whole-spot extraction, the extracts were analyzed by reversed-phase liquid chromatography-electrospray ionizationmass spectrometry in combination with the PCI-IS method. The overlaid multiple reaction monitoring (MRM) chromatograms of the PCI-IS are shown in Figure 1b. We observed a

at each retention time point. This method has been used to correct the MEs of target analytes, including pharmaceuticals and small-molecule metabolites encountered in complicated sample matrices, such as plasma or urine. In this study, we utilized the PCI-IS to determine the MEs of the first significant ion suppression zone in LC-MS, which is caused by nonretained chemicals (mainly salts) in the blood. Blood samples are isotonic and are equivalent to a 0.9% NaCl solution, and the osmotic pressure between the different blood samples should stay constant. In general, the osmotic pressure of blood is provided by the nonvolatile salts content in blood, such as sodium, potassium, and the chloride ion.26 These salts cannot be retained in reversed-phase columns, and the concentration of these salts was high. These properties of these salts cause significant ion suppression at t0 (time taken for an unretained molecule to elute from the column) in the LCESI-MS spectra, as shown in Figure 1b. Because the salt concentration of each blood sample should be similar, the total D

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

the blood volume variation on the DBS cards and the MEcaused quantification errors, it is suggested that the PCI-IS compounds selected should be structural analogs to the target analytes being quantified. Optimization of the PCI-IS Concentration and Flow Rate. The concentration and flow rate of the PCI-IS both could affect the blood volume correction efficiency. The signal intensity of the PCI-IS should be high enough to compete with the ionization of early eluting polar compounds and to generate adequate responses for observing the ion suppression change caused by different blood volumes of DBS samples. In Figure 2, higher variations were observed when the PCI-IS intensity was low. Therefore, the concentration and flow rate of the PCI-IS should be high enough to provide a stable signal. However, a higher PCI-IS flow rate would dilute the LC eluent and lead to a poor sensitivity of the target analyte. Additionally, an unnecessary high PCI-IS concentration may also affect the ionization of target analytes. Optimization of the LC Conditions. The PCI-IS measures the extent of ion suppression caused by salt in plasma samples to estimate blood volume. Therefore, the LC separation conditions should be adjusted to separate polar small-molecule compounds from salts. For reducing the number of coeluting compounds at the first significant ion suppression zone with the salts, the initial solvent strength of the mobile phase must be reduced. We observed that the initial solvent strength of 2% ACN provides satisfactory separation for salts and most polar compounds. After the nonretained chemicals were eluted, the solvent strength can be increased to improve the analytical speed. Although some polar metabolites may still coelute with salts, even at such low solvent strengths, the nonvolatile salts in blood still dominate the extent of the ion suppression of the first ion suppression zone in the PCI-IS chromatogram. The sample injection volume and the sample concentration also affected the extent of ion suppression of the PCI-IS. A too large injection volume or a too concentrated sample may decrease the difference in the extent of ion suppression caused by different blood volumes. In such a case, reducing the sample injection volume or increasing the sample dilution can increase the differences. Five microliters as the injection volume with a 10-fold sample dilution provided a good nonlinear correlation between the extent of the ion suppression and the blood volume of generally used DBS volumes. Accuracy of the PCI-IS Method for Determining Blood Volume on DBS Cards. To determine the blood volume on the DBS cards, a calibration curve generated by different blood volumes should be established. Considering the general blood volume on DBS cards, a calibration curve ranging from 5 to 25 μL of blood volume was generated. Although good correlation can be observed between the DBS volume and the minimum response at the first significant ion suppression zone (from 0.45 to 0.55 min). The volume estimation accuracy showed the best results when the calibration curve generated by the DBS volume and the reciprocal of the minimum response of PCI-IS was used. When using posaconazole as the PCI-IS with the optimum LC-MS conditions, the calibration curve generated by blood volume and the reciprocal of the minimum response of PCI-IS showed good linearity within the tested range (y = 2.2 × 10−8 x2 + 8.8 × 10−6 x + 4.4 × 10−5). Six different blood samples obtained from 6 volunteers were spotted on DBS cards, with the blood volumes between 5 and 25 μL, to test the method accuracy (n = 30). The blood spots

higher extent of ion suppression for larger blood volumes in the first ion suppression zone in the MRM chromatogram. A nonlinear regression between the extent of the ion suppression measured by the PCI-IS and the blood volume was constructed using the minimum response at the first significant ion suppression zone (from 0.45 to 0.55 min). The results indicated that the PCI-IS can effectively measure the extent of the ion suppression caused by salts in blood, and the extent of ion suppression could be used to estimate and correct the blood volume variations on DBS cards. Although blood samples collected by the collection tubes without anticoagulant could reflect DBS samples more directly, when anticoagulants are not involved, fresh blood coagulates really rapidly, which increases the difficulty of sample preparation. Therefore, the blood samples used for method development, validation, and voriconazole measurements all contain EDTA for anticoagulation. The nonlinear correlation between the DBS volume and the extent of ion suppression using DBS samples collected by the EDTA tube is shown in Figure S1. In order to obtain the best correction efficiency for the blood volume on the DBS cards, the parameters affecting the PCI-IS responses were optimized. The characteristics, the LC separation conditions, and the mass conditions of the PCI-IS compounds used all have significant effects on the correction results and were optimized accordingly. Selection of the PCI-IS. The PCI-IS method used the postcolumn infused-internal standard to measure the extent of ion suppression, and the nonlinear correlation between the extent of the ion suppression measured by the PCI-IS and the blood volume on the DBS cards determined the accuracy of the PCI-IS method for estimating blood volume. Stahnke et al.27 have indicated that MEs in ESI are not analyte dependent; MEs in ESI are primarily retention time dependent. Because PCI-IS is continuously infused through the postcolumn approach to reflect the MEs (ion suppression) at each retention time point, it does not require the use of isotope internal standards. To investigate the physical−chemical properties of the PCI-IS that are required to provide accurate estimation results, we selected 6 compounds with different functional groups and molecular weights as the test PCI-IS compounds and evaluated their ability to achieve nonlinear correlation. The structures of the tested PCI-IS compounds are shown in Figure S2. Ephedrine, flunitrazepam, butorphanol, and posaconazole were tested in the positive mode, while sulfamethoxazole was tested in the negative mode. Because HKP can be protonated in the positive mode and can also form a formic acid complex in the negative mode, HKP was detected using both positive and negative modes. Figure 2 shows the minimum response of the first ion suppression zone of the PCI-IS chromatogram of all test compounds, and it revealed a high correlation between the minimum response and the blood volume. Accordingly, we can conclude that our proposed method is very flexible in selecting an appropriate PCI-IS. The application of the PCI-IS method for the analysis of DBS cards for TDM purposes is useful not only for estimating and calibrating blood volume variations but also for correcting for ME-caused quantification errors. In our previous study, we indicated that chemicals that reflect the nonvolatile components in the matrix can provide an adjustment effect and can be used as the PCI-IS, but the adjustment effect would be further improved if the ability of the PCI-IS compound to ionize is similar to the analyte.28 Therefore, to simultaneously correct E

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

PCI-IS method was validated in terms of precision, accuracy, linearity, and sensitivity for the quantification of voriconazole on DBS cards. The effective therapeutic concentration of voriconazole is 2 μg mL−1, and the PCI-IS method in combination with the LC-ESI-MS method was validated at concentrations between 0.3, 1, and 10 μg mL−1. The pooled blood samples were spiked with voriconazole, and an equal amount (20 μL) of blood was spotted on DBS cards for the concentration measurement. The linearity was evaluated using voriconazole-spiked blood samples at the concentrations of 0.3, 1, and 10 μg mL−1. The calibration curve was y = 0.0238x − 2.7 (r2 = 0.9987). The LOD and LOQ of voriconazole on the DBS card were 100 and 300 pg mL−1, respectively (Figure S5). The intraday and interday precision of the PCI-IS method was tested four times a day for 3 days by using voriconazolespiked blood samples at concentrations of 0.3, 1, and 10 μg mL−1. The repeatability (n = 4 runs) and the intermediate precision (n = 3 days) of voriconazole at all tested concentrations were less than 14% of the relative standard deviation (RSD) (Table 2). The accuracy of the PCI-IS method was tested at concentrations of 0.3, 1, and 10 μg mL−1. The method accuracies were within 91.0% to 103.0%. The matrix effect was calculated by comparing voriconazole peak areas between standard solutions and postextraction spiked DBS samples, and the matrix effects were within 53.1% to 62.1%. The extraction recovery was tested at voriconazole concentrations of 0.3, 1, and 10 μg mL−1 with HCT values ranging from 25% to 75% (Table 3). Two hundred microliters

were extracted by methanol and were analyzed by LC-ESI-MS. The PCI-IS (posaconazole) was used to measure the extent of the ion suppression caused by the tested samples, and the extent of ion suppression was further translated into the blood volume using the preconstructed calibration curve. The test results revealed that the accuracy of the PCI-IS method was within 92.2% to 124.8% (Table 1, Figure S-3). Table 1. Blood Volume Estimation Accuracy of the PCI-IS Methoda volume (μL)

sample 1

5 10 15 20 25

124.8 92.2 96.3 93.7 93.2

a

sample 2 sample 3 sample 4 97.5 108.1 113.8 106.2 103.4

105.3 104.2 97.0 93.7 100.6

102.0 94.4 106.5 96.2 100.8

sample 5 sample 6 105.3 104.2 97.0 93.7 100.6

95.7 94.0 96.5 97.5 93.5

n = 3 runs.

Application of the PCI-IS Method for Voriconazole TDM on DBSs. One of the main advantages of using the PCIIS method for estimating the blood volume on DBS cards lies in its additional function of correcting ME-caused quantification errors. Invasive fungal infections (IFIs) are life-threatening conditions, and voriconazole is one of the main drugs used for the treatment of serious IFIs.6 TDM is important in improving the efficacy and safety of voriconazole therapy. The PCI-IS method was applied to analyze the DBS samples obtained from patients undergoing voriconazole therapy. In order to simultaneously correct the blood volume on DBS cards and calibrate the ME-caused quantification errors, posaconazole was selected as the PCI-IS. Our previous study has indicated that hydrophobicity and protonation ability are two important factors for PCI-IS selection. As both posaconazole and voriconazole are protonated in the mobile phase and they both are hydrophobic compounds, posaconazole was selected as the PCI-IS for voriconazole quantification. The concentration and flow rate of posaconazole were optimized to obtain an appropriate amount of sensitivity for the observation of the nonlinear ion suppression behaviors. The optimized concentration and flow rate of the PCI-IS were 100 ng mL−1 and 0.1 mL min−1, respectively. Mass parameters may affect the ionization efficiency and thus the extent of the ion suppression that can be detected by the PCI-IS. We found that, among four tested ESI parameters, only the drying gas temperature showed a slight effect on the correlation between the blood volume and the extent of ion suppression (Figure S-4). The minimum responses that were used to measure the extent of ion suppression increased as the drying gas temperature increased. For improved sensitivity, we selected 325 °C as the optimal drying gas temperature. Validation of the PCI-IS Method for Voriconazole TDM on DBSs. Before the analysis of the patient samples, the

Table 3. Extraction Recovery of 0.3, 1, and 10 μg mL−1 Voriconazole Spiked Blood Samples with Different Levels of HCTa

a

concentration of voriconazole (μg mL−1)

HCT 25%

HCT 50%

HCT 75%

0.3 1 10

100.1 ± 3.8 98.3 ± 3.1 105.5 ± 1.8

91.9 ± 1.3 105.5 ± 4.1 93.0 ± 0.8

108.9 ± 1.7 96.2 ± 2.8 103.4 ± 2.9

n = 4 runs.

of methanol was used for the DBS sample extraction, and the extraction recoveries for three tested concentrations in all of the HCT test ranges were within 91.9% to 108.9%. The stability was tested by storing the DBS samples at room temperature for 30 days (n = 3). The result shows that the volume estimation accuracy for DBS samples after 30 days of storage was 107 ± 7%. Correlation between the Quantification Results of DBSs and Venous Blood Sampling. Venous blood sampling is the regular approach for TDM. To evaluate the accuracy of the PCI-IS method for the quantification of voriconazole on DBS samples, we compared the quantification results obtained from venous sampling (plasma samples) and DBS sampling.

Table 2. Precision, Accuracy, and Matrix Effect of the PCI-IS for Quantifying Voriconazole in DBS Samplesa

a

concentration (μg mL−1)

accuracy (%)

matrix effect (%)

repeatability (n = 4, RSD %)

intermediate precision (n = 3, RSD %)

0.3 1 10

103.0 ± 14.3 90.9 ± 3.5 91.9 ± 7.5

53.1 ± 2.4 62.1 ± 2.7 62.1 ± 3.4

3.33 1.19 1.43

13.53 3.12 8.26

n = 3 runs. F

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

of the sample matrix, and they cause poor ionization efficiency by depressing droplet formation or evaporation or complexing with the target analytes.29 Blood salts are nonretained compounds and have strong ion suppression properties in LC-ESI-MS. Thus, the PCI-IS measures the extent of ion suppression in the first significant ion suppression zone in the chromatogram to correlate with the amount of salts in blood extracts. Polar small-molecule metabolites, such as sugars, amino acids, and organic acids, may also coelute with blood salts. Blood salts are isotonic, but the concentration of polar small-molecule metabolites may be different between individuals because of sex, diet, and health differences. To observe the effect of variation caused by differences in individual polar metabolites, we applied this PCI-IS approach to estimate the blood volumes of DBS samples collected from different subjects (n = 6), and the method accuracy was within 74.5% to 120.3%. In addition, the application of the PCI-IS method for patients undergoing voriconazole treatment also showed a high correlation between the measured results of the plasma samples and the DBS samples (r = 0.97). This result revealed that the variation between individuals of the coeluting metabolites in the first ion suppression zone has little influence on the extent of ion suppression compared to the contribution of blood salts, which is probably due to their relative amount and also their effects on ion suppression. Therefore, the PCI-IS strategy can be successfully used to reflect the blood salt content and thus the different blood volumes of DBS samples. Nevertheless, the experiments performed in this study are considered very preliminary for this promising new approach. Further investigations are required to understand the potential influences on blood components such as the albumin and lipid contents in the blood samples. Additional applications to broader groups are also required before seeing its’ true value in the clinical analysis.

Twenty-six patients undergoing voriconazole treatment were subjected to venous blood sampling and DBS sampling. We selected patients with different disease statuses including kidney failure, leukemia, meningitis, anemia, pneumonia, hepatitis, diabetes, and different types of cancer to see whether the proposed method could provide an accurate estimation for samples with diverse health conditions. The HCT values of these patients ranged from 22.4 to 38.9. The validated PCI-IS method was used to estimate and calibrate the blood volume variation after whole-spot extraction and also to quantify voriconazole concentration. Pearson’s correlation was used to evaluate the correlation between the two quantified results. The correlation coefficient of the voriconazole quantification results between the two sampling methods was 0.97 (Figure 3), and

Figure 3. Correlation of the quantification results between the DBS samples and the plasma samples obtained from 26 patients undergoing voriconazole treatment.

■

CONCLUSIONS With the growing importance of personalized medicine, dried blood sampling is considered a promising technique in advancing personalized medicine. Whole-spot extraction is advantageous because it is less affected by HCT-caused bias, but blood volume variation limits the use of this sample processing technique. This study proposed a novel approach using the PCI-IS method for estimating and correcting the blood volume variation on DBS samples. The PCI-IS method utilized the isotonic property of blood samples, which means that the total salt should positively correlate with the blood volume on DBS cards. By using a PCI-IS to measure the extent of the ion suppression at the first significant ion suppression zone in the obtained chromatograms, the blood volume on the DBS cards can be calculated and further calibrated. Because of the dual functions of PCI-IS in both calibrating blood volume variation and ME-caused quantification bias, this method is especially useful for TDM in clinical practices. We successfully applied the PCI-IS method to quantify the level of voriconazole in TDM samples, and the results showed a high correlation between DBS and conventionally used plasma samples. Although this preliminary study revealed that the PCIIS method is a simple and accurate method to calibrate blood volume variations on DBS cards, a lot more work needs to be done before it can be effectively applied in clinical settings for TDM. We anticipate the application of the PCI-IS method to diverse drugs to get more insight of the possible applications of this method.

the DBS concentration was lower than the plasma concentration. In this study, the blood samples from different patients were randomly spotted on DBS cards with volumes ranging from 5 to 15 μL to test the accuracy for blood volume estimation, and the DBS volume estimation accuracy was 108% ± 15.2%. Our results demonstrated that the PCI-IS method could accurately calibrate both the blood volume variation on the DBS cards and disease statuses and that HCT variation did not have a significant effect on estimation accuracy.

■

DISCUSSION Although DBS is considered a promising sampling technique for personalized medicine, its utilization in a clinical setting is still very limited, except with disease screening in newborns. The main hurdle of the wider application of DBS lies in its hematocrit-based bias. The isotope internal standard spraying technique was recently proposed as a way to calibrate HCTbased bias,9,14 but this approach will increase the cost of the DBS method. It is well recognized that whole spot extraction is less affected by HCT-based bias, but controlling for the blood volume became its main challenge. In this study, the PCI-IS method was demonstrated to be an effective method for estimating and calibrating blood volume variations on DBS cards. This method used a PCI-IS to measure the total salts in the blood extracts and further translate this measure into blood volume. The nonvolatile salts in the blood are one of the main causes of the ion suppression G

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

■

(21) Gonzalez, O.; van Vliet, M.; Damen, C. W. N.; van der Kloet, F. M.; Vreeken, R. J.; Hankemeier, T. Anal. Chem. 2015, 87, 5921−5929. (22) Lord, R. C. C. Postgrad. Med. J. 1999, 75, 67−73. (23) Spanakis, E. K.; Aperis, G.; Mylonakis, E. Clin. Infect. Dis. 2006, 43, 1060−1068. (24) Desta, Z.; Zhao, X. J.; Shin, J. G.; Flockhart, D. A. Clin. Pharmacokinet. 2002, 41, 913−958. (25) Pascual, A.; Calandra, T.; Bolay, S.; Buclin, T.; Bille, J.; Marchetti, O. Clin. Infect. Dis. 2008, 46, 201−211. (26) Prosser, C. L.; Green, J. W.; Chow, T. J. Biol. Bull. 1955, 109, 99−107. (27) Stahnke, H.; Reemtsma, T.; Alder, L. Anal. Chem. 2009, 81, 2185−2192. (28) Chen, G. Y.; Liao, H. W.; Tseng, Y. J.; Tsai, I. L.; Kuo, C. H. Anal. Chim. Acta 2015, 864, 21−29. (29) King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942−950.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01145. Additional figures including the minimum response in the first inhibition zone of different volume DBSs obtained from blood samples, chemical structures, blood volume estimation accuracy, the influence of the mass ionization parameters, and chromatograms; table of precision of blood volume estimation (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +886.2.33668766. Fax: +886.2.23919098. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by the Ministry of Science and Technology of Taiwan (MOST 104-2113-M-002-009-). The authors thank the NTU Integrated Core Facility for Functional Genomics of National Research Program for Genomic Medicine of Taiwan for their technical assistance.

■

REFERENCES

(1) Wilhelm, A. J.; den Burger, J. C. G.; Swart, E. L. Clin. Pharmacokinet. 2014, 53, 961−973. (2) Martin, N. J.; Cooper, H. J. Expert Rev. Proteomics 2014, 11, 685− 695. (3) Li, W. K.; Tse, F. L. S. Biomed. Chromatogr. 2010, 24, 49−65. (4) Robijns, K.; Koster, R. A.; Touw, D. J. Clin. Pharmacokinet. 2014, 53, 1053−1053. (5) ter Heine, R.; Beijnen, J. H.; Huitema, A. D. R. Bioanalysis 2009, 1, 1329−1338. (6) Barfield, M.; Spooner, N.; Lad, R.; Parry, S.; Fowles, S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 870, 32−37. (7) Spooner, N.; Lad, R.; Barfield, M. Anal. Chem. 2009, 81, 1557− 1563. (8) Ramakrishnan, Y.; Shabbir, S.; Miller, S.; Houlden, J.; Shadare, J.; Dewit, O.; Barfield, M.; Spooner, N. Br. J. Clin. Pharmacol. 2009, 68, 274−306. (9) Abu-Rabie, P.; Denniff, P.; Spooner, N.; Chowdhry, B. Z.; Pullen, F. S. Anal. Chem. 2015, 87, 4996−5003. (10) Burnett, J. E. C. Bioanalysis 2011, 3, 1099−1107. (11) Oliveira, R. V.; Henion, J.; Wickremsinhe, E. R. Bioanalysis 2014, 6, 2027−2041. (12) Li, F. M.; Zulkoski, J.; Fast, D.; Michael, S. Bioanalysis 2011, 3, 2321−2333. (13) Denniff, P.; Spooner, N. Bioanalysis 2010, 2, 1385−1395. (14) Abu-Rabie, P.; Denniff, P.; Spooner, N.; Brynjolffssen, J.; Galluzzo, P.; Sanders, G. Anal. Chem. 2011, 83, 8779−8786. (15) Edelbroek, P. M.; van der Heijden, J.; Stolk, L. M. L. Ther. Drug Monit. 2009, 31, 327−336. (16) Choi, B. K.; Gusev, A. I.; Hercules, D. M. Anal. Chem. 1999, 71, 4107−4110. (17) Cheng, C.; Tsai, H. R. Anal. Chim. Acta 2008, 623, 168−177. (18) Liao, H. W.; Chen, G. Y.; Wu, M. S.; Liao, W. C.; Tsai, I. L.; Kuo, C. H. J. Chromatogr A 2015, 1375, 62−68. (19) Liao, H. W.; Chen, G. Y.; Tsai, I. L.; Kuo, C. H. J. Chromatogr A 2014, 1327, 97−104. (20) Liao, H. W.; Tsai, I. L.; Chen, G. Y.; Lu, Y. S.; Lin, C. H.; Kuo, C. H. J. Chromatogr A 2014, 1358, 85−92. H

DOI: 10.1021/acs.analchem.6b01145 Anal. Chem. XXXX, XXX, XXX−XXX