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Ning Sun Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Zhuo Li Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China
School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, China

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Meng Zhang Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Huan He Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Libo Zhao Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Dong Mei Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Guanghua Zhu Hematopoietic Stem Cell Transplantation Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Xiaoling Wang Pharmaceutical Department, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China

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Abstract

In this work, a simple and rapid high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to carry out the simultaneous measurement of busulfan (BU) and phenytoin (PHT) in the plasma of children. In this method, plasma sample could be prepared by one-step protein precipitation using 1 mL of methanol/water (1:1, v/v). After centrifugation (14,500 rpm, 5 min, 4 °C), 10 μL of the supernatant was injected into a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm, Thermo Fisher Scientific) for separation by gradient elution. Quantification was carried out using multiple reactions monitoring (MRM) under positive scan mode. In the method verification, the calibration curves of BU and PHT showed satisfactory linearity (r > 0.99) at the concentration ranging from 0.02 to 20 μg mL−1. The accuracy and precision were tested at four concentration levels (including the LLOQ level) with the relative error (RE) ranging from −0.80% to 11.45% and coefficient of variation (CV) between 0.93% and 7.74%. There was no pronounced matrix effect to interfere with the quantitative analysis. Compared to determine BU and PHT using two individual methods, less pre-treatment process, labor and blood sample volume are required in this proposed method. Finally, this method was successfully applied to the therapeutic drug monitoring of BU and PHT for children underwent hematological stem cell transplantation.

Abstract

In this work, a simple and rapid high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to carry out the simultaneous measurement of busulfan (BU) and phenytoin (PHT) in the plasma of children. In this method, plasma sample could be prepared by one-step protein precipitation using 1 mL of methanol/water (1:1, v/v). After centrifugation (14,500 rpm, 5 min, 4 °C), 10 μL of the supernatant was injected into a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm, Thermo Fisher Scientific) for separation by gradient elution. Quantification was carried out using multiple reactions monitoring (MRM) under positive scan mode. In the method verification, the calibration curves of BU and PHT showed satisfactory linearity (r > 0.99) at the concentration ranging from 0.02 to 20 μg mL−1. The accuracy and precision were tested at four concentration levels (including the LLOQ level) with the relative error (RE) ranging from −0.80% to 11.45% and coefficient of variation (CV) between 0.93% and 7.74%. There was no pronounced matrix effect to interfere with the quantitative analysis. Compared to determine BU and PHT using two individual methods, less pre-treatment process, labor and blood sample volume are required in this proposed method. Finally, this method was successfully applied to the therapeutic drug monitoring of BU and PHT for children underwent hematological stem cell transplantation.

1 Introduction

Busulfan (1, 4-butanediol dimethansulfonate, BU), as a DNA alkylating agent to suppress cell division, is widely used for myeloablative preconditioning regimens before hematological stem cell transplantation (HSCT) [1]. It has been fully aware that BU has a narrow therapeutic range and shows a noticeable inter-individual difference [2, 3]. The BU exposure, expressed as area under the concentration-time curve (AUC), is critical for prognosis after HSCT. Specifically, high BU exposure (AUC > 1,350 μmol·min·L−1) means an increased risk for venous-occlusive disease and even transplant-related mortality. On the contrary, low exposure (AUC < 900 μmol·min·L−1) is associated with graft-rejection or disease relapse [3]. Therefore, the personalized dose adjustment through therapeutic drug monitoring (TDM) for BU is essential for children with HSCT.

In the preconditioning regimens of HSCT, 16 doses of BU intravenous administration in a 4 days' routine period according to the dosage of 2–4 mg/kg/day are the most commonly scheme [4]. BU could allodially cross the blood-brain barrier and cause high levels in the central nervous system, which may cause the onset of seizures [5]. According the previous reports, the incidence of BU-induced seizures is about 10% when no any precautionary measures were used [6]. Therefore, antiepileptic drugs (AEDs) have been used to prevent seizures during the BU conditioning. Phenytoin (PHT), as the most commonly used AEDs, has been reported to lower the seizure rates to 0–5.5% [5]. But there were also some defects for PHT, such as nonlinear pharmacokinetics, uncertain bioavailability (20–100%), and obvious inter-individual difference. For the past few years, levetiracetam, as the second-generation AED, has been developed and its effectiveness in the prevention of seizures also has been proven [7], meanwhile, it shows better pharmacokinetic characteristics with a bioavailability nearly to 100%. However, its safety and efficacy data in young children (under the age of four) is lacking, which limited its extensive application in BU conditioning. Up to now, PHT is still the most widely used applied AED. Therefore, it is necessary and meaningful to carry out the individualized medication of PHT base on TDM.

Now, TDM for BU and PHT were carried out separately in pediatric clinic. PHT is usually administrated orally every time at the beginning of intravenous infusion of BU. Almost the same dosing time means the TDM of BU and PHT could use a same blood sampling scheme, which might avoid repeated blood sampling and reduces the pain of the children. Meanwhile, developing a method to simultaneously assay the both drugs would save labor costs and time, and improve the efficiency of TDM.

Currently, the assay of PHT in clinic was mainly performed by immunoassay method [8]. For BU assay, there were lots of methods have been reported like enzyme linked immunosorbent assay (ELISA) [9], high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) [10] and HPLC-MS/MS method [11]. However, no method has been reported that could simultaneously measure both drugs until now.

The purpose of the present work was to develop and validate a HPLC-MS/MS method to carry out the simultaneous determination of BU and PHT in the plasma sample of children. In this method, a small sample volume (50 μL of plasma) could be rapidly prepared by one-step protein precipitation method without any dilution operation. Only five minutes of instrumental analytical time was needed for one sample. Compared to separately assay the both drugs, plenty of pre-treatment processes, labor and blood sample volume could be saved. Finally, the proposed method was successfully applied to the simultaneous TDM of BU and PHT for children undergoing HSCT.

2 Materials and methods

2.1 Chemicals and reagents

Standard of BU was purchased from Sigma (St. Louis, MO, USA). The d8-labeled BU (BU-d8) was purchased from Bioruler (Danbury, CT, USA). Standard of PHT was purchased from National Institutes for Food and Drug Control (Beijing, China). Meanwhile, HPLC-grade methanol was purchased from MREDA (Beijing, China). Other chemicals used were analytical or HPLC grade. Distilled water used in this work was purchased from Watsons (Hong Kong, China). Blank human plasma samples were supported by Beijing Children's Hospital.

2.2 Conditions of LC-MS/MS

The sample analysis was performed by a LC-20 chromatography system (Shimadzu, Kyoto, Japan) coupled to an QTRAP 5500 triple quadrupole tandem mass spectrometry (Sciex, Toronto, Canada) equipped with a Turbo Ion Spray Source operating in positive ion mode. Quantification was carried out by multiple reactions monitoring (MRM) mode. The needle potential and the source temperature were set at 5500 V and 550 °C, respectively. The other parameters, including declustering potential (DP), collision cell exit potential (CXP), entrance potential (EP), and collision energy (CE), have been optimized (Table 1). The chromatographic separation was performed on a Hypersil Gold C18 column (150 × 2.1 mm, 5 μm) (Thermo Scientific, USA) by gradient elution with the mobile phase (A) 2 mM ammonium acetate containing 0.4% formic acid and (B) methanol. The gradient elution program was: 0–0.5 min, 40% B; 0.5–1.5 min, 40–85% B; 1.5–3.5 min, 85% B; 3.5–3.6 min, 85–40% B; 3.6–5.0 min, 40% B. The total flow rate was 0.4 mL min−1.

Table 1.

MRM transitions for quantification of BU, PHT and BU-d8 (IS)

Analyte Precursor ion (m/z) Product ions (m/z) DP EP CE CXP
BU 264.0 151.0 50 15 16 8
PHT 253.0 182.0 80 15 23 15
BU-d8 272.0 159.0 50 15 16 8

Abbreviations: MRM, multiple reaction monitoring; BU, busulfan; PHT, phenytoin; IS, internal standard; m/z, mass-to-charge ratio; DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.

2.3 Standard solutions

The standard solutions of BU and PHT were prepared by dissolution in methanol/water (1:1, v/v). Mixed working standard solutions were prepared by serial dilution of standard solutions with methanol/water (1:1, v/v) to obtain solutions of 0.05, 0.1, 0.25, 2.5, 12.5, 25.0, 37.5, 50 μg mL−1 for BU and PHT. The quality control solutions were prepared as described above and the quality control working solutions were prepared by serial dilution to obtain solutions of 0.1, 12.5, 37.5 μg mL−1. The prepared standard and quality control solutions were stored at −40 °C, and working solutions were freshly prepared prior to use.

Bu-d8, as the internal standard (IS) compound of BU and PHT, was weighed and dissolved in methanol/water (1:1, v/v) at 1.0 mg mL−1, then the IS working solution was prepared by diluting with methanol/water (1:1, v/v) to a concentration of 1.0 μg mL−1. The IS working solution was divided into aliquots and stored at −40 °C with single aliquots thawed prior to analysis.

2.4 Sample preparation

Plasma (50 μL) was mixed with 20 μL of the IS solution. Methanol/water (1:1, v/v) (1 mL) was added to precipitate proteins, and mixed by vortexing for 30 s. The precipitate was removed by centrifugation for 5 min at 14,500 rpm at 4 °C. The supernatant (10 μL) was injected into the LC-MS/MS for analysis.

2.5 Method application

Clinical samples were collected from six patients who underwent HSCT in Beijing Children's Hospital. This study was approved by the ethics committee in hospital. BU began to administrate from 8 d before transplantation by intravenous infusion (2 h of infusion, every 6 h) for a period of 3–4 d. Meanwhile, PHT was administrated orally every time at the beginning of intravenous infusion of BU. Blood sample (0.4 mL) was collected into EDTA tubes at 0.5, 1, 2, 2.5, 4, 6 h after the first dose and centrifuged at 3,500 rpm for 10 min at 4 °C to yield plasma supernatants. Then, the plasma samples were immediately analyzed by the proposed method. The AUCs of the six patients were calculated by Phoenix WinNonlin 8.1 software.

3 Results and discussion

3.1 MS condition optimization

The mass spectrometric conditions of BU, PHT and IS have been optimized respectively by infusing standard solutions into mass spectrometer using syringe infusion pump. In positive mode, BU and IS (BU-d8) could produce sufficient precursor ions of [M+NH4]+ ions: 264.0 (m/z) and 272.0 (m/z), respectively. PHT could stably obtain [M+H]+ ions of 253.0 (m/z). Furthermore, 151 (m/z), 159 (m/z) and 182 (m/z) could be obtained as the most abundant product ions of BU, BU-d8 and PHT. So these MRM transitions above have been selected for quantification analysis. The chemical constructions and full scan product ions of precursor ions of BU, PHT and BU-d8 were shown in Fig. 1.

Fig. 1.
Fig. 1.

The chemical structures and full scan product ion of precursor ions of BU, PHT and their IS (BU-d8)

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01069

3.2 HPLC condition optimization

The clear difference between the chemical construction of BU and PHT means different chromatographic behaviors, which required detailed condition optimization to get better peak pattern, higher detection signal, and the shortest possible run time. During the method development, different types of chromatographic column and mobile phase have been tested to achieve this purpose. In consideration of weak polarity of the two drugs, the convention C18 sorbent might provide enough adsorption capacity, therefore, two sizes of Hypersil Gold C18 column (150 mm × 4.6/2.1 mm, 5 μm, Thermo Scientific, USA) have been tested, and the smaller diameter column (150 mm × 2.1 mm, 5 μm) shows better retention for two drugs than the larger one. Then, the gradient elution mode has been selected according to the test, because the isocratic elution could not meet the needs of both appropriate retention and fast elution. Subsequently, the mobile phase has been optimized in detail. The result of the investigation shows methanol could acquire better peak shapes than acetonitrile. Meanwhile, ammonium acetate added to the aqueous phase could improve the signal of PHT but lower the signal of BU. Then, the gradient elution program and the concentration of ammonium acetate have been further optimized. Finally, 2 mM ammonium acetate containing 0.4% formic acid and methanol as the mobile phase of gradient elution were used to chromatographic separation. The representative chromatograms were shown in Fig. 2 with no interference found at the retention time of analyzes and IS.

Fig. 2.
Fig. 2.

Representative chromatograms of BU (I), PHT (II) and IS (III) in plasma sample. (A) Blank plasma sample; (B) Blank plasma spiked with IS; (C) Blank plasma spiked with BU (0.02 μg mL−1), PHT (0.02 μg mL−1) and IS; (D) Plasma sample collected from subject of NO. 3 (25 min) after dosing of BU and PHT

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01069

3.3 Linearity

The calibration curve samples were prepared by using 20 μL of standard solutions to replace 20 μL of methanol/water (1:1, v/v) to obtain plasma concentrations of 0.02, 0.04, 0.1, 1, 5, 10, 15, 20 μg mL−1. The calibration curve for BU and PHT were constructed respectively by plotting the peak area ratios of analytes/IS using weighted least square linear regression analysis with 1/X2 as the weighting factor. The calibration curves were constructed and validated thrice in different batches with good linearity (r > 0.99) achieved.

3.4 Precision and accuracy

Quality control samples have been prepared according to the description above to evaluate the precision and accuracy of the method. Six replicates were analyzed at four concentration levels (including the LLOQ level) in three days. Inter/intra-day coefficient of variation (CV) and relative error (RE), expressed as precision and accuracy respectively, are shown in Table 2. The result shows the CVs ranged from 0.93% from 7.74%, and REs were between −0.80 and 11.45%.

Table 2.

The accuracy and precision of the methods (n = 6)

Analyte Spiked (μg mL−1) Intra-day Inter-day
Mean found (μg mL−1) RE (%) CV (%) Mean found (μg mL−1) RE (%) CV (%)
BU 0.02 0.0212 6.00 6.04 0.0210 5.15 7.24
0.04 0.0427 6.70 0.93 0.0415 3.73 2.76
5 5.33 6.56 2.17 5.22 4.49 2.28
15 15.36 2.40 1.57 14.88 −0.80 2.83
PHT 0.02 0.0223 11.50 3.56 0.0217 8.42 7.16
0.04 0.0446 11.45 4.94 0.0420 4.95 7.16
5 5.42 8.44 1.78 5.24 4.75 5.66
15 16.48 9.87 1.45 15.1 0.09 7.74

Abbreviations: BU, busulfan; PHT, phenytoin; RE, relative error; CV, coefficient of variation.

3.5 Recovery

In this work, a recovery test was carried out to assess the loss of sample preparation procedure. The recovery test samples were prepared by adding three levels standard solutions into the blank post-extraction matrix homogenate. The peak areas of BU, PHT, and IS measured in recovery test samples were considered as theoretical values, and the relative peak area measured in quality control samples were considered as tested values. The ratio of the test values to theoretical values were evaluated as the recovery. Six replicates were performed for each concentration level. The results are shown in Table 3. The average recovery of BU, PHT, and IS was 74.49–85.20%, 90.12–93.40%, and 72.53%, respectively.

Table 3.

Extraction recoveries of the target and IS compounds (n = 6)

Analyte Spiked (ng mL−1) Recovery (%) IS Recovery (%)
BU 0.04 74.49 72.53
5 76.17
15 85.20
PHT 0.04 92.91
5 90.12
15 93.40

Abbreviations: BU, busulfan; PHT, phenytoin; IS, internal standard.

3.6 Matrix effect

Matrix effect is an important issue in biological sample analysis, especially to a LC-MS/MS method. So, the matrix effects of BU, PHT and IS in plasma samples were evaluated in this work, respectively. Standards of two drugs and IS was added into both blank solvents and the post-extraction plasma matrix. The blank plasma used was collected from six different sources. The ratio of peak area measured in the plasma matrix sample to the corresponding in pure solvent was defined as the matrix factor (MF). The MF ratio of analyte to IS was defined as the IS-normalized MF. The results are shown in Table 4. It could be found that the CVs of IS-normalized MF for both BU and PHT were less than 2.07% and 3.93%, which means the interference from plasma matrix is slight to the quantitative analysis. Meanwhile, the result met the requirements (≤15%) of relevant guiding principles for biological sample analysis.

Table 4.

Matrix effects of plasma samples (n = 6)

Analyte Spiked (μg mL−1) IS-normalized MF CV (%)
BU 0.04 1.05 2.07
5 1.07 0.97
15 1.12 2.06
PHT 0.04 3.03 3.93
5 2.99 2.00
15 3.08 3.54

Abbreviations: BU, busulfan; PHT, phenytoin; IS, internal standard; MF, matrix factor; CV, coefficient of variation.

3.7 Stability

It is vital to the whole TDM work to ensure the stability of drugs in sample matrix. In this work, blood samples collected from the patients after dosing of BU and PHT were used to assess the stability of the drugs in whole blood and plasma sample under several different conditions. The concentration differences between the sample measured immediately and samples stored under different conditions prior to measurement were defined as variable coefficient (VC).
V C ( % ) = value ( measured immediately” ) value ( stored under different conditions ) value ( measured immediately ) × 100 %

The result listed in Table 5 shows no obvious differences between the measured value of immediately prepared whole blood sample and these stored at 4 °C for 3 h with the VC for BU and PHT were −6.80% and −6.32%, respectively. Meanwhile, in Table 6, the measured value of plasma samples prepared immediately were consistent with these stored at room temperature (24 °C) for 3 h, stored at 4 °C for 24 h and stored at −40 °C for 24 h with the VC for BU ranged from −5.02 to −0.24%, and VC for PHT ranged from −4.17 to 0.69%, respectively. Furthermore, the stability of drugs in hemolytic plasma was also assessed by assaying the spiked (0.04 and 15 μg mL−1) hemolytic plasma samples, which stored at 4 °C for 3 h. The result shows the CVs ranged from 2.46% from 6.44%, and REs were between −6.58 and 3.25% (n = 3).

Table 5.

Stability of BU and PHT in whole blood sample of patient

Analyte Measured immediately (μg mL−1) After stored at 4 °C for 3 h (μg mL−1) VC (%)
BU 0.815 0.760 −6.80
PHT 4.59 4.30 −6.32

Abbreviations: BU, busulfan; PHT, phenytoin; VC, variable coefficient.

Table 6.

Stability of BU and PHT in plasma sample of patient

Analyte Measured immediately (μg mL−1) After stored at 24 °C for 3 h (μg mL−1) VC (%) After stored at 4 °C for 24 h (μg mL−1) VC (%) After stored at −40 °C for 24 h (μg mL−1) VC (%)
BU 0.837 0.811 −3.11 0.835 −0.24 0.795 −5.02
PHT 14.40 14.40 0 14.50 0.69 13.80 −4.17

Abbreviations: BU, busulfan; PHT, phenytoin; VC, variable coefficient.

3.8 Method application

The BU and PHT plasma concentrations of the six patients were measured by the established method. The plasma concentrations-time curves were shown in Fig. 3. The basic information of patients and the TDM result of BU and PHT were listed in Table 7. There was no any interfering peak observed in the determination of six children, which indicated the high selectivity of this method. From the result, almost all patients' AUCs of BU failed to reach the recommend range (900–1,350 μmol·min·L−1). Meanwhile, the PHT level of several patients might be insufficient according to the recommend concentration range (10–20 μg mL−1) [5]. Sample preparation and analysis could be finished within 2 h include eight calibration curves samples, three quality control samples, and six collected samples from patient. High efficiency of the proposed method helps to rapidly acquire the result of concentration or AUC, and timely to implement the dosing adjustment.

Fig. 3.
Fig. 3.

BU and PHT plasma concentration-time curves of the six patients

Citation: Acta Chromatographica 2022; 10.1556/1326.2022.01069

Table 7.

The basic information of the six patients and their TDM results of BU and PHT

NO. Sex Age Weight (kg) Dose of BU (mg) Dose of PHT (mg) AUC of BU (μmol·min·L−1) Level range of PHT (μg mL−1)
1 female 4 years 15 18 37.5 831.41 1.75–4.95
2 male 9 years 30 27 70 820.18 10.50–18.10
3 female 12 years 80 50 100 776.65 3.35–5.77
4 female 6 years 18 20 50 562.67 5.80–12.50
5 female 11 years 38.5 30 75 633.98 5.37–10.30
6 male 10 years 54 43 100 1,098.40 8.10–20.30

Abbreviations: TDM, therapeutic drug monitoring; BU, busulfan; PHT, phenytoin; AUC, area under the concentration-time curve.

3.9 Method comparison and discussion

This work summarizes different methods for assay both PHT and BU in biological sample, including immunoassay, HPLC, GC-MS and HPLC-MS/MS (Table 8). Clearly, the concentration of BU or PHT could be quickly measured by an immunoassay [8] or an ELISA method [9]. Still, the inevitable cross-reaction always limits its accuracy and sensitivity. Sample pre-processing like liquid-liquid extraction or solid-phase extraction were time consuming, so these operations in GC-MS [12, 13] and HPLC-UV [10] method may not match the need for quick detection for TDM, especially for BU, its UV absorption only occurs after the derivatization. Therefore, HPLC–MS/MS method [14–16] seems to be the most widely used in BU detection, the reason may be tedious derivatization step has been eliminated and no any cross-reaction existed.

Table 8.

Analytical methods applied for the determination of BU and PHT

Year Analyte Analytical methodology Extraction methodology and characteristics Analytical measurement range (μg mL−1) Analytical run time (min) References
2005 PHT Turbidimetric Immunoassay Immunoadsorption 0.79–46.1 <1 [8]
2013 PHT GC-MS Solid-phase extraction 0.05–1.2 30 [12]
2017 PHT HPLC Dispersive liquid-phase microextraction 0.002–0.4 7 [17]
2018 PHT HPLC-MS/MS Protein precipitation 0.01–2.0 7 [14]
1999 BU HPLC Liquid-liquid extraction followed by derivatization and re-extracted 0.05–2.0 30 [10]
2009 BU ELISA Immunoadsorption 0.075–2.0 <1 [9]
2012 BU GC-MS Liquid-liquid extraction followed by derivatization, evaporation, and reconstitution 0.04–0.4 5 [13]
2014 BU HPLC-MS/MS Protein precipitation, dilution of supernatant 0.06–2.0 4 [15]
2020 BU HPLC-MS/MS Protein precipitation 0.01–1.0 5 [16]
The present work BU, PHT HPLC-MS/MS Protein precipitation 0.02–20.0 5 \

Abbreviations: BU, busulfan; PHT, phenytoin; GC-MS, gas chromatography–mass spectrometry; HPLC, high performance liquid chromatography; HPLC-MS/MS, high performance liquid chromatography-mass spectrometry; ELISA, enzyme linked immunosorbent assay.

As the primary extraction methodology in the reported HPLC-MS/MS methods, protein precipitation is convenient and straightforward for plasma sample preparation before analysis. In our previous work [16], pure organic solvents, as protein precipitants, were found could not achieve ideal signal response of BU than a mixture of organic solvents and water, because the pure organic solvents like acetonitrile or methanol may allow protein to clump together quickly and wrap the analytes and IS as co-precipitation which reduces their concentration in supernatant. The addition of water can prevent protein from clumping together and improve drug dispersion in solution. Therefore, in this work, 1 mL of mixture of methanol and water (1:1, v/v) was used as protein precipitant after investigation and optimization of the ratio of the two phases. So, this one-step protein precipitation also could be considered as a combination of precipitation and dilution operation. In this way, the processing time and labor could be significantly saved.

Until now, there was no a HPLC-MS/MS method has been reported to simultaneously measure the concentrations of the both drugs. Compared with the previous work, the TDM of the two drugs could be simultaneously carried out without adding any additional blood collection. It could be considered as a children patient friendly attempt. In addition, less blood collection, but more clinical benefit may be a promising research direction in pediatric TDM.

4 Conclusions

A rapid and simple HPLC-MS/MS method was developed and validated for the simultaneous measurement of BU and PHT in human plasma. One-step protein precipitation was needed in the sample preparation without any dilution procedure. A single HPLC-MS/MS run for two drugs could be rapidly achieved within five minutes. Compared with using two individual methods to measure BU and PHT, less pre-treatment process, labor and blood sample volume were required in this method. The point is that the established method was successfully applied to the simultaneous TDM of BU and PHT for children underwent HSCT.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This work was supported by Capital Medical University basic clinical research cooperation project in 2017 (No.17JL08) and Major projects of the Ministry of Science and Technology (No. 2018ZX09721003).

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • Á. M. Móricz (Centre for Agricultural Research, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

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2021  
Web of Science  
Total Cites
WoS
652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
Impact Factor
without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
Journal Citation Indicator 0,40
Rank by Journal Citation Indicator Chemistry, Analytical 72/99
Scimago  
Scimago
H-index
29
Scimago
Journal Rank
0,27
Scimago Quartile Score Chemistry (miscellaneous) (Q3)
Scopus  
Scopus
Cite Score
2,8
Scopus
CIte Score Rank
General Chemistry 210/409 (Q3)
Scopus
SNIP
0,586

2020
 
Total Cites
650
WoS
Journal
Impact Factor
1,639
Rank by
Chemistry, Analytical 71/83 (Q4)
Impact Factor
 
Impact Factor
1,412
without
Journal Self Cites
5 Year
1,301
Impact Factor
Journal
0,34
Citation Indicator
 
Rank by Journal
Chemistry, Analytical 75/93 (Q4)
Citation Indicator
 
Citable
45
Items
Total
43
Articles
Total
2
Reviews
Scimago
28
H-index
Scimago
0,316
Journal Rank
Scimago
Chemistry (miscellaneous) Q3
Quartile Score
 
Scopus
393/181=2,2
Scite Score
 
Scopus
General Chemistry 215/398 (Q3)
Scite Score Rank
 
Scopus
0,560
SNIP
 
Days from
58
submission
 
to acceptance
 
Days from
68
acceptance
 
to publication
 
Acceptance
51%
Rate

2019  
Total Cites
WoS
495
Impact Factor 1,418
Impact Factor
without
Journal Self Cites
1,374
5 Year
Impact Factor
0,936
Immediacy
Index
0,460
Citable
Items
50
Total
Articles
50
Total
Reviews
0
Cited
Half-Life
6,2
Citing
Half-Life
8,3
Eigenfactor
Score
0,00048
Article Influence
Score
0,164
% Articles
in
Citable Items
100,00
Normalized
Eigenfactor
0,05895
Average
IF
Percentile
20,349
Scimago
H-index
26
Scimago
Journal Rank
0,255
Scopus
Scite Score
226/167=1,4
Scopus
Scite Score Rank
Chemistry (miscellaneous) 240/398 (Q3)
Scopus
SNIP
0,494
Acceptance
Rate
41%

 

Acta Chromatographica
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Acta Chromatographica
Language English
Size A4
Year of
Foundation
1992
Volumes
per Year
1
Issues
per Year
4
Founder Institute of Chemistry, University of Silesia
Founder's
Address
PL-40-007 Katowice, Poland, Bankowa 12
Publisher Akadémiai Kiadó
Publisher's
Address
H-1117 Budapest, Hungary 1516 Budapest, PO Box 245.
Responsible
Publisher
Chief Executive Officer, Akadémiai Kiadó
ISSN 2083-5736 (Online)

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