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MS – Two case examples
Journal of Pharmaceutical and Biomedical Analysis 183 (2020) 113135
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS – Two case examples Guohua An a,∗ , Thanh Bach a , Inas Abdallah a,b , Demet Nalbant a a b
Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa USA Analytical Chemistry Department, Faculty of Pharmacy, University of Sadat City, Egypt
a r t i c l e
i n f o
Article history: Received 22 November 2019 Received in revised form 3 January 2020 Accepted 27 January 2020 Available online 30 January 2020 Keywords: LC/MS/MS Matrix effect Analyte effect Phospholipid monitoring Electrospray ionization Ion suppression
a b s t r a c t The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: the first compromise is made between sample cleanliness and sample preparation speed since protein precipitation does not provide very clean final extract; the second compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has to be accepted. The first compromise may lead to matrix effect, which is caused by co-eluting endogenous substances such as phospholipids. The second compromise can result in analyte effect, which is caused by co-eluting analyte(s). We have encountered the issue of matrix/analyte-mediated ion suppression in multiple preclinical and clinical pharmacokinetic projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we presented two real case examples on signal suppression caused by either endogenous phospholipids or co-eluting analyte. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a powerful quantitative analytical technique that has been widely used in many clinical and research laboratories. Compared with traditional high performance liquid chromatography (HPLC), LC/MS/MS provides better specificity because of its ability to monitor selected mass ions and better sensitivity due to the enhanced signal-to-noise ratio. Another advantage of LC/MS/MS is that it can measure multiple analytes even when they are eluted together (i.e. chromatographic peak overlap) because LC/MS/MS identify and monitor analytes based on their molecular weight (molecular ion) and structure (product ion). This is different from HPLC in which complete chromatographic peak separation is required and consequently long sample run time is often needed in order to separate the peaks. Because of this,
∗ Corresponding author at: Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, 115 S Grand Ave, Iowa City, IA, 52242, USA. E-mail address: [email protected] (G. An). https://doi.org/10.1016/j.jpba.2020.113135 0731-7085/© 2020 Elsevier B.V. All rights reserved.
LC/MS/MS offers short sample run time and represents a particularly powerful tool for multi-analyte measurement in complicated matrix (e.g. biological samples). Because of the specificity and selectivity achieved with MS analyzers, a natural thought is that chromatographic separation can be minimized or even eliminated for LC/MS/MS. This is a misconception as co-eluting endogenous substances (i.e. matrix component) or compounds, although in general will not interfere with mass ion detection, could significantly affect the efficiency and reproducibility of the ionization process [1,2]. These result in signal suppression or, in rare cases, signal enhancement, which is termed matrix effect (for co-eluting matrix component) or analyte effect (for co-eluting analyte(s)) [3]. Both matrix effect and analyte effect can affect the quantitative performance of the LC/MS/MS and lead to unreliable results [4]. Electrospray ionization (ESI) is known to be more prone to ion suppression than atmospheric pressure chemical ionization (APCI) [5]. The origin of the signal suppression lies in the ESI mechanism itself and can be ascribed to the competition among ion species for the limited number of charged surface sites present on the generated droplets during the electrospray process [1]. We have encountered the issue of matrix/analyte-mediated ion suppression repetitively in multiple preclinical and clinical pharmacokinetic
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(PK) projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we present two real case examples of the matrix/analyte-mediated ion suppression. 2. Methods 2.1. Chemicals and reagents Piperacillin sodium salt, tazobactam, cefazolin, ampicillin, and sulbactam, were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2 H5 -Piperacilline was purchased from Alsachim Lab (Strasbourg, France). Acetonitrile, methanol, water, and formic acid, all of which were LC/MS grade, were purchased from Fisher Scientific (Fairlawn, NJ, USA). Human plasma with lithium heparin were purchased from BioreclamationIVT (Westbury, NY, USA). All chemicals were of the highest purity available from commercial providers. 2.2. Instrumentation Both projects presented in this paper were done using the same LC/MS/MS instrument, which includes Shimadzu UFLC20AD system (Shimadzu, Japan) and an API4000 triple quadrupole mass spectrometer (AB Sciex LLC, Redwood City, CA, USA). Shimadzu UFLC-20AD system consists of an LC-20AD binary pump, a SIL-20ACHT UFLC autosampler, a CTO-20A column oven, and a DGU-20A3R degasser. All compounds were detected using a TurboIonSpray® probe via multiple reaction monitoring (MRM) by the API4000 triple quadrupole mass spectrometer. The Instrument Control and Data Processing Software Analyst 1.6.2 (AB Sciex LLC, Redwood City, CA, USA) was used for data acquisition and processing. 2.3. Analytical conditions Project 1 (CAS project) The LC column used in the project was Phenomenex SynergiTM C18 column (150 x 20 mm, 4 m) coupled with a Phenomenex 4 x 2 mm security guard cartridge (both from Phenomenex, Torrance, CA, USA). The mobile phase consisted of a mixture of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Several gradient conditions were tested during assay development. Originally, mobile phase was delivered under a total flow rate of 0.3 mL/min; the gradient was started with 10% B and increased to 95% B from 0.3 to 1.0 min, maintained at 95% B until 4.0 min, and held at 10% from 4.0 to 6.0 min. In the final LC condition, the total flow rate was 0.35 mL/min and the gradient used was as follows: 5% B (0–0.5 min), 5–35% B (0.5–1.5 min), 55% B (2.5–4.5 min), and 5% B (5.7–7.5 min). The typical retention time of cefazolin, ampicillin and sulbactam under these gradient conditions were presented in Table 1. The MRM transitions for the antibiotics and their respective internal standard (i.e., IS) with the corresponding compound specific parameters are shown in Table 2. Project 2 (PTMC project) Phenomenex Kinetex® C18 100 Å LC column (50 x 2.1 mm, 2.6 m) coupled with a Phenomenex SecurityGuard ULTRA cartridge UPLC Evo C18 (both from Phenomenex, Torrance, CA, USA) was used in this assay. The mobile phase used for analysis was a mixture of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B), which were combined in a gradient as follows: 2% B (0-0.3 min), 40% B (0.3–2.8 min), and 2% B (2.8 min until the end of the run). The mobile phase was
Table 1 Two LC conditions for CAS assay and the retention time of analytes under each condition. Original LC condition
New LC condition
Total flow rate LC gradient
0.3 mL/min 0.0 – 0.3 min: 10 %B 0.3 – 1.0 min: 10 – 95 %B 1.0 – 4.0 min: 95 %B 4.0 – 6.0 min: 10 %B
Total run time Cefazolin retention time Sulbactam retention time Ampicillin retention time 2 H5 -piperacillin retention time
6.0 min 3.57 min 3.57 min 3.58 min 3.68 min
0.35 mL/min 0.0 – 0.5 min: 5 %B 0.5 – 1.5 min: 5 – 35 %B 1.5 – 2.5 min: 35 %B 2.5 – 4.5 min: 35 – 55 %B 4.5 – 5.7 min: 55 – 5 %B 5.7 – 7.5 min: 5 %B 7.5 min 4.54 min 3.97 min 4.02 min 5.82 min
delivered at a total flow rate of 0.25 mL/min. The total run time was 7.0 min. Under these conditions, piperacillin and tazobactam had typical retention times of 3.45 and 2.75 min, respectively. 2 H5 piperacillin, IS for piperacillin and tazobactam, eluted with typical retention time of 3.42 min. The MRM transitions for the antibiotics and their respective IS with the corresponding compound specific parameters are shown in Table 2. 2.4. Preparation of calibration standards and quality control samples Project 1 (CAS project) Stock solution of cefazolin, sulbactam, ampicillin and 2 H5 -piperacillin (i.e. IS) was prepared in methanol at concentrations of 6 mg/mL, 6 mg/mL, 3 mg/mL and 1 mg/mL, respectively. All stock solutions were stored at −80 ◦ C. Working solution of cefazolin, sulbactam and ampicillin were prepared by serial dilution with methanol. Working solution of 2 H5 -piperacillin was prepared by serial dilution with methanol to a final concentration of 25 g/mL. Calibration standards and quality control (QC) samples were prepared by spiking 90 L of blank human plasma with 10 L of the appropriate CAS working solution and 10 L of IS working solution followed by brief vortex. Blank sample (i.e. no analyte) was prepared by spiking 90 L of blank human plasma with 10 L of methanol and 10 L of IS working solution; and double blank sample (i.e. no analyte, no IS) was prepared by adding 20 L of methanol to 90 L of blank human plasma. The final concentration of IS in plasma was 2.5 g/mL. Table 3 presents the final concentration of CAS in calibration standards as well as QC samples. Project 2 (PTMC project) Stock solutions of antibiotics and 2 H5 piperacillin (IS) were prepared by dissolving compounds in methanol for a concentration of 2 mg/mL and 1 mg/mL, respectively. All stock solutions were stored at -20 ◦ C. Working solutions for piperacillin and tazobactam were prepared by serial dilution in methanol. Calibration standards and QC samples for all tested antibiotics were prepared by spiking appropriate working solution to blank plasma with lithium heparin, and exact concentrations are provided in Table 3. IS working solutions were also prepared in a similar way. Final concentration of IS in each sample was 2.5 g/mL. 2.5. Sample preparation Project 1 (CAS project) All calibration standards and QC samples were extracted using protein precipitation with 4 volumes of acetonitrile. After sample centrifugation at 17,000 g and 4 ◦ C for 20 min, 20 L of supernatant were further diluted with 100 L of water and transferred to LC/MS vials for analysis. Project 2 (PTMC project) Samples were vortexed and 4 volumes of acetonitrile was added to each sample for protein precipitation. After further vortexing, the samples were centrifuged at 17,000 g
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Table 2 Mass spectrometry parameters for PTMC and CAS assays. Project
PTMC
CAS
Ionization mode
Positive
Compound
Corresponding IS
Precursor ion m/z (Q1)
Product ion m/z (Q3)
DP (V)
CE (V)
CXP (V)
Source parameters
Cefepime Meropenem Piperacillin Tazobactam 2 H6 -meropenem 2 H5 -piperacillin Cefazolin
2
H5 -piperacillin
Sulbactam
2
H5 -piperacillin
234.3
Ampicillin
2
H5 -piperacillin
350.3
2
H5 -piperacillin
545.3 453.3
Sulbactam
2
H5 -piperacillin
232.5
Ampicillin
2
H5 -piperacillin
348.2
396.1 141.1 398.1 168.1 147.1 398.1 323.4 295.3 142.0 192.1 160.2 192.1 398.1 320.7 251.4 141.2 189.3 207.4 270.4 331.1
40.0 40.0 40.0 40.0 40.0 40.0 30.0 30.0 30.0 60.0 60.0 60.0 60.0 −30.0 −30.0 −30.0 −30.0 −30.0 −100.0 −30.0
15.0 20.0 20.0 20.0 20.0 20.0 25.0 25.0 25.0 25.0 35.0 35.0 35.0 −10.0 −15.0 −18.0 −15.0 −18.0 −18.0 −18.0
12.0 12.0 12.0 12.0 12.0 12.0 11.0 15.0 8.0 8.0 11.0 11.0 11.0 −8.0 −15.0 −14.0 −14.0 −12.0 −8.0 −8.0
CAD: 9 psig CUR: 10 psig GS1: 50 psig GS2: 40 psig IS: 5000 V TEMP: 500 ◦ C
2
481.4 384.3 540.3 301.4 390.3 545.3 455.0
Positive
2 H5 -piperacillin Cefazolin
CAS
H6 -meropenem H6 -meropenem 2 H5 -piperacillin 2 H5 -piperacillin 2
Negative
2
H5 -piperacillin
521.8
CAD: 9 psig CUR: 10 psig GS1: 50 psig GS2: 40 psig IS: 5000 V TEMP: 500 ◦ C CAD: 10 CUR: 30 GS1: 50 GS2: 50 IS: -4500 V TEMP: 500 ◦ C
DP: declustering potential; CE: collision energy; CXP: cell exit potential; CAD: collision gas; CUR: curtain gas; GS1: gas 1 (nebulizer gas); GS2; gas 2 (turbo gas); IS: ionspray voltage; TEMP: temperature. Entrance potential (EP) was 10.0 V in positive ionization mode and -10.0 V in negative ionization mode.
Table 3 Concentration of calibration standards and quality control (QC) samples of PTMC and CAS (LLOQ: lower limit of quantification, QC med: QC medium). PTMC project Calibrator 1 Calibrator 2 Calibrator 3 Calibrator 4 Calibrator 5 Calibrator 6 Calibrator 7 Calibrator 8 Calibrator 9 QC LLOQ QC low QC med QC high
Cefepime(g/mL) 0.5 1 5 10 25 50 100 150 – 0.5 1.5 75 120
CAS project Meropenem(g/mL) 0.1 0.25 0.5 1 5 10 50 100 150 0.1 0.3 75 120
Piperacillin(g/mL) 0.1 0.5 1 5 10 50 100 150 – 0.1 0.3 75 120
for 15 min at 4 ◦ C. Following this, 10 L of the supernatant of each sample was diluted with 90 L of water. The contents were then transferred to LC/MS vials for analysis. 3. Results 3.1. [CAS project, matrix effect] Ion suppression of analyte due to co-eluting endogenous phospholipids The overall goal of this project is to evaluate the PK of cefazolin, ampicillin and sulbactam in patients undergoing surgery. To achieve this goal, one necessary step is to establish bioanalytical method(s) to quantify these antibiotics in human plasma. We developed a LC/MS/MS assay for quantification of cefazolin, ampicillin and sulbactam simultaneously. During assay development phase, the lower limit of quantification (i.e. LLOQ) of cefazolin was found to be 0.5 g/mL, with the signal to noise ratio being >50. However, during assay validation phase, both inter-day and intraday accuracy and precision of the LLOQ of cefazolin at 0.5 g/mL failed (i.e. out of the 20% acceptance criteria) in multiple runs, indicating that the signal was unstable. In addition, at the LLOQ level, the peak shape of cefazolin was poor, with peak shoulders shown in some but not all LLOQ calibration samples as well as some but not all analytical runs. We terminated the assay validation and went back
Tazobactam(g/mL) 0.25 0.5 1 5 10 50 100 150 – 0.25 0.75 75 120
Cefazolin(g/mL) 0.25 0.5 1 5 10 50 150 200 300 0.25 0.75 75 240
Sulbactam(g/mL) 0.5 1 5 10 20 50 100 150 200 0.5 1.5 75 180
Ampicillin(g/mL) 0.5 1 5 10 20 50 100 150 200 0.5 1.5 75 180
to assay development process. We suspected that the poor peak shape and unstable LLOQ of cefazolin may be caused by endogenous substances in matrix (i.e. human plasma). A simple protein precipitation method was used during the sample preparation process. This procedure can remove proteins but cannot efficiently eliminate lipids. Phospholipids are the main component of the cell membranes and glycerophosphocholines (PCs), including 2-lyso PC and diradyl PC, are the major class of phospholipids in the plasma (Fig. 1). To identify the impact of PCs on the elution and signal of cefazolin, we added a transition of m/z 184 > 184 to monitor PCs. The transition of 184 > 184 was used because both 2-lyso PC and diradyl PC are known to undergo in source collision-induced dissociation (CID) and generate fragment with same m/z ratio of 184, which allows for monitoring all PCs in one transition (i.e. 184 > 184). As shown in Fig. 2a, under the original LC gradient condition, cefazolin in a LLOQ calibration sample was eluted at 3.59 min with a clear shoulder on its peak. In addition, PCs were co-eluted at the same time with more than 100-fold higher peak intensity. To avoid the interference with PCs, a new LC gradient condition was used to separate cefazolin and PCs. As shown in Fig. 2b, under this new LC gradient condition, cefazolin and PCs were eluted at different time. As a result, peak shape, peak height, and peak intensity of cefazolin were all greatly improved. With this optimized condition, cefazolin LLOQ was reduced from 0.5 g/mL to 0.25 g/mL, assay
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Fig. 1. Structure of phospholipids. The glycerophosphocholines (PCs), the major class of phospholipids in the plasma, can be divided based on their different glycerin groups into 2-lyso PC (left) and diradyl PC (right).
validation was performed and all inter-day and intra-day accuracy and precision of cefazolin were successfully passed at the LLOQ level. As noted earlier, under the original assay condition, the peak shape of cefazolin was different among different analytical runs, and peak shoulder was observed in some but not all LLOQ samples. Since there are many different PCs, although they have same fragment and can be monitored together in one channel, they could be eluted at different times. We anticipate that the poor peak shape of cefazolin is due to the interference with different PCs, and the magnitude of interference may vary from sample to sample depending on the level of the different PCs. To test this hypothesis, blank human plasma from 6 different lots were processed using same protein precipitation method, and the elution patterns as well as intensity of PCs among these samples were compared. As shown in Fig. 3a, we found that multiple PC peaks were eluted with variable intensities. In our current assay, the protein precipitation method was performed using acetonitrile. Since methanol is also a common choice for protein precipitation, a natural question is that which solvent is more efficient in removing phospholipids in plasma. To address this question, a number of blank human plasma samples were precipitated with either acetonitrile or methanol and then the PCs in these samples were compared. As shown in Fig. 3, compared with samples extracted by acetonitrile (Fig. 3a), similar intensity of PCs were detected in samples extracted by methanol (Fig. 3b). Therefore, under the sample extraction procedure used in our study, acetonitrile is not more efficient in removing phospholipids in plasma than methanol. 3.2. [PTMC project, analyte effect] Ion suppression of stable-isotope-labeled internal standard due to co-eluting unlabeled analyte The overall goal of this project was to evaluate the PK of piperacillin, tazobactam, meropenem, and cefepime in patients from intensive care unit or patients with cystic fibrosis. We have successfully developed and fully validated a robust and sensitive LC/MS/MS assay for the quantification of these four antibiotics simultaneously. All the work was done under the GLP environment and we have reported the details of the assay validation result recently [6,7]. In this assay, 2 H5 -piperacillin was used as the IS for piperacillin and tazobactam, and 2 H6 -meropenem was the IS for meropenem and cefepime. Matrix/analyte effect was not observed during assay development or validation phases. However, this issue came up when clinical samples containing piperacillin and tazobactam were analyzed. Fig. 4 shows the typical chromatograms of piperacillin, tazobactam as well as 2 H5 -piperacillin in one calibration sample. Both piperacillin and tazobactam have same upper limit of quantification (i.e. ULOQ) of 150 g/mL. In one of the analytical runs, among 60 clinical samples analyzed, the cal-
culated concentrations of piperacillin in 17 samples were above the ULOQ and the calculated concentrations of tazobactam in all samples were within the ULOQ. The standard procedure is to accept tazobactam results, reject those piperacillin results outside of the calibration range, and rerun the samples after appropriate dilution for piperacillin quantification. However, after further data evaluation we noticed that the peak area of 2 H5 -piperacillin in those clinical samples with piperacillin higher than ULOQ was substantially lower than that in the calibration samples or clinical samples with piperacillin within ULOQ level (Fig. 5). The phenomenon was consistently observed in different batches which included those clinical samples with piperacillin concentrations higher than ULOQ. The most likely explanation for this phenomenon is ion suppression of 2 H5 -piperacillin when it was co-eluting with much higher concentration of piperacillin. Since 2 H5 -piperacillin was used as the IS of tazobactam, the abnormally low peak area of 2 H5 -piperacillin resulted in unreliable tazobactam quantification. The calculated tazobactam concentrations in those samples were much higher than the real concentrations. After careful examination, we rejected both piperacillin and tazobactam results obtained from clinical samples with piperacillin concentrations higher than ULOQ, and reran those samples with appropriate dilution. 4. Discussion In this paper, we presented two case examples on signal suppression caused by either endogenous phospholipids (matrix effect, example 1) or co-eluting analyte (analyte effect, example 2). Although the “syndrome” of these signal suppression is different, they are caused by the same fundamental mechanism and can be explained by Enke’s model for electrospray response [1]. During electrospray ionization process, droplets with a surface excess charge are created. As shown in Enke’s model, there is a limit on the number of excess charge sites available on the surface. As a result, competition for ionization can occur when more than one ionic species are eluted from the LC column at the same time and move to the ESI probe together [1]. It has been reported that under conditions commonly seen in LC/MS/MS for small-molecule compounds (e.g. flow rates > 0.1 mL/min; relatively high concentrations such as 10−6 to 10-4 M in the bulk phase), the total concentration of excess charge (Q) is typically close to 10-9 eq/L [8]. At such values of Q, the competition between analytes with abundant endogenous substances or other analytes becomes evident. During the process of competition for excess charge sites, between the two co-eluted ionic species, the one with much lower concentration will be affected more and consequently have more significant ion suppression. In case example 1, a matrix effect from co-eluting phospholipids was observed. The consequence of this matrix effect is the poor peak shape and unstable LLOQ of the co-eluting analyte (i.e. cefazolin) as well as the corresponding decreased assay sensitivity.
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Fig. 2. Representative LC/MS/MS chromatograms of phospholipids and cefazolin under a) old LC gradient condition and b) new LC gradient condition. The MRM transitions for phospholipids and cefazolin were m/z 184.0 > 184.0 and 453.3 > 320.7, respectively. LC gradient condition is expressed as % of acetonitrile.
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Fig. 3. LC/MS/MS chromatograms of phospholipids from samples prepared by protein precipitation method using a) acetonitrile or b) methanol. Samples were prepared from 6 different lots of human plasma with lithium heparin. LC gradient condition is expressed as % of acetonitrile.
Fig. 4. Representative LC/MS/MS chromatograms for piperacillin, tazobactam, as well as their internal standard 2 H5 -piperacillin. The MRM transitions for piperacillin, tazobactam, and 2 H5 -piperacillin were m/z 540.3 > 398.1, 301.4 > 168.1, and 545.3 > 398.1, respectively.
Our observation is consistent with literature reports. Phospholipids have been reported to interfere with many compounds and have been considered as the major cause of matrix effect [9,10]. Chambers et al. monitored the levels of various phospholipids in the samples to compare the relative cleanliness of final plasma extract from different sample preparation procedures [11]. They reported that protein precipitation is the least effective sample preparation method, while the mixed-mode strong cation exchange solid phase extraction (SPE) is the most effective method in eliminating phospholipids [11]. In addition, single step liquid-liquid extraction (LLE) with MTBE yields extracts of comparable cleanliness to mixed mode cation SPE [11]. To overcome the phospholipids-mediated ion suppression, there are following two main strategies: 1) To improve the cleanliness of final plasma extract by switching protein precipitation method to more efficient LLE or SPE. However, as both LLE and SPE are time consuming and labor intensive, this strategy may not be suitable for the situation where high throughput sample analysis is required. Although protein precipitation does not provide very clean final extract, its advantage of simple, fast and less labor intensive allows for high throughput sample analysis. In our projects, high throughput sample analysis is required as thousands
Fig. 5. Peak area of piperacillin (circles) and 2 H5 -piperacillin (triangles) in calibration standards as well as 17 clinical samples. Final concentration of 2 H5 -piperacillin spiked in each sample was 2.5 g/mL. Although same concentration of 2 H5 -piperacillin was spiked in each sample, the peak area of 2 H5 -piperacillin in those 17 piperacillin clinical samples (right panel) was substantially lower than that in piperacillin calibration standard samples (left panel). For those 17 clinical samples, the concentrations of piperacillin were all higher than the ULOQ (i.e. 150 g/mL).
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of clinical samples need to be analyzed. In this case, the strategy of switching protein precipitation method to LLE or SPE is not preferred. 2) To optimize the LC condition to separate the analyte peak and phospholipids peaks. This is the strategy we preferred and used in our project since it does not conflict with high throughput sample analysis. Considering that phospholipids represent the major source of matrix effect, adding the transition of 184 > 184 to monitor PCs will be highly valuable and should be considered during assay development phase. In addition, using different ionization method such as APCI may also avoid significant matrix effect since APCI is known to be less prone to ion suppression than ESI. In case example 2, an analyte effect on stable-isotope labeled analyte was observed and it was caused by the co-eluting unlabeled analyte. In the current example, this analyte effect was manifested as a significant decrease of the internal standard (2 H5 -piperacillin) peak area when the concentration of the unlabeled analyte (i.e. piperacillin) in clinical samples exceeded a threshold. It should be noted that this analyte effect on 2 H5 -piperacillin may not have significant impact on the calculated concentration of piperacillin as the ratio of piperacillin and 2 H5 -piperacillin signals should still be relatively proportional to the ratio of piperacillin’s concentrations. However, this analyte effect can become a serious problem for multi-analyte procedures in which the stable-isotope labeled internal standard is not only used as internal standard for the respective unlabeled analogue, but also for other analytes, which is the exact situation that we encountered. As piperacillin-mediated ion suppression on 2 H5 -piperacillin did not occur in calibration standard samples and only happened in those clinical samples containing piperacillin higher than 150 g/mL, this analyte effect can be easily overlooked and consequently substantial quantification bias on tazobactam would result. Therefore, for such multi-analyte procedures, it is important to carefully examine the peak area of the stable-isotope-labeled internal standard before accepting the calculated concentrations of the other analytes. In case study 2, we showed an example of ion suppression on internal standard by the co-eluting high concentration of analyte. Theoretically, there could be a mutual interaction between the analyte and co-eluting internal standard. This means that if the concentration of the internal standard is high, it potentially could cause the ion suppression of the co-eluting analyte. This mutual suppression between analyte and co-eluting internal standard has been reported by Sojo et al., and they commented that co-eluting internal standard could have substantial impact on the method detection limits [8]. In their study, the IS concentrationdependent ion suppression clearly shown, indicating that selection of the concentration of the internal standard is of importance [8]. Traditionally, the concentration of the internal standards is usually selected to be midway of the calibration range. This approach may cause significant ion suppression of the analyte in those calibration standard samples containing low concentrations of analyte, resulting in increase in detection limit (i.e. decreased sensitivity). Different strategies may be needed depending on the type of internal standard. If the internal standard is a different compound compared with the analyte, mobile phase condition should be optimized to ensure that IS and the analyte are not eluted at the same time. If peak separation is not successful, different internal standard should be considered. If the internal standard used in the method is stable-isotope-labeled analyte, peak separation between IS (labelled analyte) and analyte itself is impossible. In this case, the concentration of labeled IS must be selected carefully with the goal of maintaining the desired detection limit of the analyte. The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: one compromise is made between sample cleanliness
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and sample preparation speed since it is known that protein precipitation does not provide very clean final extract [12]; another compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has be to accepted. It should keep in mind that matrix effect may occur when the final sample extract is not clean and analyte effect may occur when there is overlap or co-elution of some peaks. Therefore, during assay development process, it is necessary to add a channel to monitor phospholipids and make sure that they are not eluted at the same time with the analyte(s). During assay validation process, it is important to evaluate not only matrix effect but also the interference between analytes and ISs. The evaluation of the interference between analytes and ISs is particularly important when they are co-eluted, such as isotope-labeled IS where peak separation is not possible. As noted earlier, the concentration of labeled IS must be selected carefully to make sure that the LLOQ of the analtye is not significantly affected. Similarly, the effect of analyte at the ULOQ level on the co-eluting IS should also be evaluated to make sure that the peak area of the IS is not significantly reduced. Author statement Guohua An: Conceptualization, methodology, Writing- Original draft preparation, Funding acquisition Thanh Bach: methodology, experiment and data collection, Manuscript review and editing Inas Abdallah: experiment and data collection Demet Nalbant: experiment and data collection Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Partial support was provided by the Division of Microbiology and Infectious Disease, the National Institute of Allergy and Infectious Disease, the National Institutes of Health (HHSN272200800008C) References [1] C.G. Enke, A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes, Anal. Chem. 69 (23) (1997) 4885–4893. [2] T.M. Annesley, Ion suppression in mass spectrometry, Clin. Chem. 49 (7) (2003) 1041–1044. [3] F. Gosetti, E. Mazzucco, D. Zampieri, M.C. Gennaro, Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry, J. Chromatogr. A 1217 (25) (2010) 3929–3937. [4] F.T. Peters, D. Remane, Aspects of matrix effects in applications of liquid chromatography-mass spectrometry to forensic and clinical toxicology–a review, Anal. Bioanal. Chem. 403 (8) (2012) 2155–2172. [5] C. Ghosh, C.P. Shinde, B.S. Chakraborty, Influence of ionization source design on matrix effects during LC-ESI-MS/MS analysis, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 893-894 (2012) 193–200. [6] R. D’Cunha, T. Bach, B.A. Young, P. Li, D. Nalbant, J. Zhang, P. Winokur, G. An, Quantification of Cefepime, Meropenem, Piperacillin, and tazobactam in human plasma using a sensitive and robust liquid chromatography-tandem mass spectrometry method, part 1: assay development and validation, Antimicrob. Agents Chemother. 62 (9) (2018). [7] R. D’Cunha, T. Bach, B.A. Young, P. Li, D. Nalbant, J. Zhang, P. Winokur, G. An, Quantification of Cefepime, Meropenem, Piperacillin, and tazobactam in human plasma using a sensitive and robust liquid chromatography-tandem mass spectrometry method, part 2: stability evaluation, Antimicrob. Agents Chemother. 62 (9) (2018). [8] L.E. Sojo, G. Lum, P. Chee, Internal standard signal suppression by co-eluting analyte in isotope dilution LC-ESI-MS, Analyst 128 (1) (2003) 51–54. [9] O.A. Ismaiel, M.S. Halquist, M.Y. Elmamly, A. Shalaby, H.T. Karnes, Monitoring phospholipids for assessment of matrix effects in a liquid
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chromatography-tandem mass spectrometry method for hydrocodone and pseudoephedrine in human plasma, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 859 (1) (2007) 84–93. [10] J.H. Ye, L.H. Pao, Using visualized matrix effects to develop and improve LC-MS/MS bioanalytical methods, taking TRAM-34 as an example, PLoS One 10 (4) (2015), e0118818.
[11] E. Chambers, D.M. Wagrowski-Diehl, Z. Lu, J.R. Mazzeo, Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 852 (1-2) (2007) 22–34. [12] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds, Rapid Commun. Mass Spectrom. 13 (12) (1999) 1175–1185.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS – Two case examples Guohua An a,∗ , Thanh Bach a , Inas Abdallah a,b , Demet Nalbant a a b
Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa USA Analytical Chemistry Department, Faculty of Pharmacy, University of Sadat City, Egypt
a r t i c l e
i n f o
Article history: Received 22 November 2019 Received in revised form 3 January 2020 Accepted 27 January 2020 Available online 30 January 2020 Keywords: LC/MS/MS Matrix effect Analyte effect Phospholipid monitoring Electrospray ionization Ion suppression
a b s t r a c t The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: the first compromise is made between sample cleanliness and sample preparation speed since protein precipitation does not provide very clean final extract; the second compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has to be accepted. The first compromise may lead to matrix effect, which is caused by co-eluting endogenous substances such as phospholipids. The second compromise can result in analyte effect, which is caused by co-eluting analyte(s). We have encountered the issue of matrix/analyte-mediated ion suppression in multiple preclinical and clinical pharmacokinetic projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we presented two real case examples on signal suppression caused by either endogenous phospholipids or co-eluting analyte. © 2020 Elsevier B.V. All rights reserved.
1. Introduction Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a powerful quantitative analytical technique that has been widely used in many clinical and research laboratories. Compared with traditional high performance liquid chromatography (HPLC), LC/MS/MS provides better specificity because of its ability to monitor selected mass ions and better sensitivity due to the enhanced signal-to-noise ratio. Another advantage of LC/MS/MS is that it can measure multiple analytes even when they are eluted together (i.e. chromatographic peak overlap) because LC/MS/MS identify and monitor analytes based on their molecular weight (molecular ion) and structure (product ion). This is different from HPLC in which complete chromatographic peak separation is required and consequently long sample run time is often needed in order to separate the peaks. Because of this,
∗ Corresponding author at: Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, 115 S Grand Ave, Iowa City, IA, 52242, USA. E-mail address: [email protected] (G. An). https://doi.org/10.1016/j.jpba.2020.113135 0731-7085/© 2020 Elsevier B.V. All rights reserved.
LC/MS/MS offers short sample run time and represents a particularly powerful tool for multi-analyte measurement in complicated matrix (e.g. biological samples). Because of the specificity and selectivity achieved with MS analyzers, a natural thought is that chromatographic separation can be minimized or even eliminated for LC/MS/MS. This is a misconception as co-eluting endogenous substances (i.e. matrix component) or compounds, although in general will not interfere with mass ion detection, could significantly affect the efficiency and reproducibility of the ionization process [1,2]. These result in signal suppression or, in rare cases, signal enhancement, which is termed matrix effect (for co-eluting matrix component) or analyte effect (for co-eluting analyte(s)) [3]. Both matrix effect and analyte effect can affect the quantitative performance of the LC/MS/MS and lead to unreliable results [4]. Electrospray ionization (ESI) is known to be more prone to ion suppression than atmospheric pressure chemical ionization (APCI) [5]. The origin of the signal suppression lies in the ESI mechanism itself and can be ascribed to the competition among ion species for the limited number of charged surface sites present on the generated droplets during the electrospray process [1]. We have encountered the issue of matrix/analyte-mediated ion suppression repetitively in multiple preclinical and clinical pharmacokinetic
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(PK) projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we present two real case examples of the matrix/analyte-mediated ion suppression. 2. Methods 2.1. Chemicals and reagents Piperacillin sodium salt, tazobactam, cefazolin, ampicillin, and sulbactam, were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2 H5 -Piperacilline was purchased from Alsachim Lab (Strasbourg, France). Acetonitrile, methanol, water, and formic acid, all of which were LC/MS grade, were purchased from Fisher Scientific (Fairlawn, NJ, USA). Human plasma with lithium heparin were purchased from BioreclamationIVT (Westbury, NY, USA). All chemicals were of the highest purity available from commercial providers. 2.2. Instrumentation Both projects presented in this paper were done using the same LC/MS/MS instrument, which includes Shimadzu UFLC20AD system (Shimadzu, Japan) and an API4000 triple quadrupole mass spectrometer (AB Sciex LLC, Redwood City, CA, USA). Shimadzu UFLC-20AD system consists of an LC-20AD binary pump, a SIL-20ACHT UFLC autosampler, a CTO-20A column oven, and a DGU-20A3R degasser. All compounds were detected using a TurboIonSpray® probe via multiple reaction monitoring (MRM) by the API4000 triple quadrupole mass spectrometer. The Instrument Control and Data Processing Software Analyst 1.6.2 (AB Sciex LLC, Redwood City, CA, USA) was used for data acquisition and processing. 2.3. Analytical conditions Project 1 (CAS project) The LC column used in the project was Phenomenex SynergiTM C18 column (150 x 20 mm, 4 m) coupled with a Phenomenex 4 x 2 mm security guard cartridge (both from Phenomenex, Torrance, CA, USA). The mobile phase consisted of a mixture of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Several gradient conditions were tested during assay development. Originally, mobile phase was delivered under a total flow rate of 0.3 mL/min; the gradient was started with 10% B and increased to 95% B from 0.3 to 1.0 min, maintained at 95% B until 4.0 min, and held at 10% from 4.0 to 6.0 min. In the final LC condition, the total flow rate was 0.35 mL/min and the gradient used was as follows: 5% B (0–0.5 min), 5–35% B (0.5–1.5 min), 55% B (2.5–4.5 min), and 5% B (5.7–7.5 min). The typical retention time of cefazolin, ampicillin and sulbactam under these gradient conditions were presented in Table 1. The MRM transitions for the antibiotics and their respective internal standard (i.e., IS) with the corresponding compound specific parameters are shown in Table 2. Project 2 (PTMC project) Phenomenex Kinetex® C18 100 Å LC column (50 x 2.1 mm, 2.6 m) coupled with a Phenomenex SecurityGuard ULTRA cartridge UPLC Evo C18 (both from Phenomenex, Torrance, CA, USA) was used in this assay. The mobile phase used for analysis was a mixture of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B), which were combined in a gradient as follows: 2% B (0-0.3 min), 40% B (0.3–2.8 min), and 2% B (2.8 min until the end of the run). The mobile phase was
Table 1 Two LC conditions for CAS assay and the retention time of analytes under each condition. Original LC condition
New LC condition
Total flow rate LC gradient
0.3 mL/min 0.0 – 0.3 min: 10 %B 0.3 – 1.0 min: 10 – 95 %B 1.0 – 4.0 min: 95 %B 4.0 – 6.0 min: 10 %B
Total run time Cefazolin retention time Sulbactam retention time Ampicillin retention time 2 H5 -piperacillin retention time
6.0 min 3.57 min 3.57 min 3.58 min 3.68 min
0.35 mL/min 0.0 – 0.5 min: 5 %B 0.5 – 1.5 min: 5 – 35 %B 1.5 – 2.5 min: 35 %B 2.5 – 4.5 min: 35 – 55 %B 4.5 – 5.7 min: 55 – 5 %B 5.7 – 7.5 min: 5 %B 7.5 min 4.54 min 3.97 min 4.02 min 5.82 min
delivered at a total flow rate of 0.25 mL/min. The total run time was 7.0 min. Under these conditions, piperacillin and tazobactam had typical retention times of 3.45 and 2.75 min, respectively. 2 H5 piperacillin, IS for piperacillin and tazobactam, eluted with typical retention time of 3.42 min. The MRM transitions for the antibiotics and their respective IS with the corresponding compound specific parameters are shown in Table 2. 2.4. Preparation of calibration standards and quality control samples Project 1 (CAS project) Stock solution of cefazolin, sulbactam, ampicillin and 2 H5 -piperacillin (i.e. IS) was prepared in methanol at concentrations of 6 mg/mL, 6 mg/mL, 3 mg/mL and 1 mg/mL, respectively. All stock solutions were stored at −80 ◦ C. Working solution of cefazolin, sulbactam and ampicillin were prepared by serial dilution with methanol. Working solution of 2 H5 -piperacillin was prepared by serial dilution with methanol to a final concentration of 25 g/mL. Calibration standards and quality control (QC) samples were prepared by spiking 90 L of blank human plasma with 10 L of the appropriate CAS working solution and 10 L of IS working solution followed by brief vortex. Blank sample (i.e. no analyte) was prepared by spiking 90 L of blank human plasma with 10 L of methanol and 10 L of IS working solution; and double blank sample (i.e. no analyte, no IS) was prepared by adding 20 L of methanol to 90 L of blank human plasma. The final concentration of IS in plasma was 2.5 g/mL. Table 3 presents the final concentration of CAS in calibration standards as well as QC samples. Project 2 (PTMC project) Stock solutions of antibiotics and 2 H5 piperacillin (IS) were prepared by dissolving compounds in methanol for a concentration of 2 mg/mL and 1 mg/mL, respectively. All stock solutions were stored at -20 ◦ C. Working solutions for piperacillin and tazobactam were prepared by serial dilution in methanol. Calibration standards and QC samples for all tested antibiotics were prepared by spiking appropriate working solution to blank plasma with lithium heparin, and exact concentrations are provided in Table 3. IS working solutions were also prepared in a similar way. Final concentration of IS in each sample was 2.5 g/mL. 2.5. Sample preparation Project 1 (CAS project) All calibration standards and QC samples were extracted using protein precipitation with 4 volumes of acetonitrile. After sample centrifugation at 17,000 g and 4 ◦ C for 20 min, 20 L of supernatant were further diluted with 100 L of water and transferred to LC/MS vials for analysis. Project 2 (PTMC project) Samples were vortexed and 4 volumes of acetonitrile was added to each sample for protein precipitation. After further vortexing, the samples were centrifuged at 17,000 g
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Table 2 Mass spectrometry parameters for PTMC and CAS assays. Project
PTMC
CAS
Ionization mode
Positive
Compound
Corresponding IS
Precursor ion m/z (Q1)
Product ion m/z (Q3)
DP (V)
CE (V)
CXP (V)
Source parameters
Cefepime Meropenem Piperacillin Tazobactam 2 H6 -meropenem 2 H5 -piperacillin Cefazolin
2
H5 -piperacillin
Sulbactam
2
H5 -piperacillin
234.3
Ampicillin
2
H5 -piperacillin
350.3
2
H5 -piperacillin
545.3 453.3
Sulbactam
2
H5 -piperacillin
232.5
Ampicillin
2
H5 -piperacillin
348.2
396.1 141.1 398.1 168.1 147.1 398.1 323.4 295.3 142.0 192.1 160.2 192.1 398.1 320.7 251.4 141.2 189.3 207.4 270.4 331.1
40.0 40.0 40.0 40.0 40.0 40.0 30.0 30.0 30.0 60.0 60.0 60.0 60.0 −30.0 −30.0 −30.0 −30.0 −30.0 −100.0 −30.0
15.0 20.0 20.0 20.0 20.0 20.0 25.0 25.0 25.0 25.0 35.0 35.0 35.0 −10.0 −15.0 −18.0 −15.0 −18.0 −18.0 −18.0
12.0 12.0 12.0 12.0 12.0 12.0 11.0 15.0 8.0 8.0 11.0 11.0 11.0 −8.0 −15.0 −14.0 −14.0 −12.0 −8.0 −8.0
CAD: 9 psig CUR: 10 psig GS1: 50 psig GS2: 40 psig IS: 5000 V TEMP: 500 ◦ C
2
481.4 384.3 540.3 301.4 390.3 545.3 455.0
Positive
2 H5 -piperacillin Cefazolin
CAS
H6 -meropenem H6 -meropenem 2 H5 -piperacillin 2 H5 -piperacillin 2
Negative
2
H5 -piperacillin
521.8
CAD: 9 psig CUR: 10 psig GS1: 50 psig GS2: 40 psig IS: 5000 V TEMP: 500 ◦ C CAD: 10 CUR: 30 GS1: 50 GS2: 50 IS: -4500 V TEMP: 500 ◦ C
DP: declustering potential; CE: collision energy; CXP: cell exit potential; CAD: collision gas; CUR: curtain gas; GS1: gas 1 (nebulizer gas); GS2; gas 2 (turbo gas); IS: ionspray voltage; TEMP: temperature. Entrance potential (EP) was 10.0 V in positive ionization mode and -10.0 V in negative ionization mode.
Table 3 Concentration of calibration standards and quality control (QC) samples of PTMC and CAS (LLOQ: lower limit of quantification, QC med: QC medium). PTMC project Calibrator 1 Calibrator 2 Calibrator 3 Calibrator 4 Calibrator 5 Calibrator 6 Calibrator 7 Calibrator 8 Calibrator 9 QC LLOQ QC low QC med QC high
Cefepime(g/mL) 0.5 1 5 10 25 50 100 150 – 0.5 1.5 75 120
CAS project Meropenem(g/mL) 0.1 0.25 0.5 1 5 10 50 100 150 0.1 0.3 75 120
Piperacillin(g/mL) 0.1 0.5 1 5 10 50 100 150 – 0.1 0.3 75 120
for 15 min at 4 ◦ C. Following this, 10 L of the supernatant of each sample was diluted with 90 L of water. The contents were then transferred to LC/MS vials for analysis. 3. Results 3.1. [CAS project, matrix effect] Ion suppression of analyte due to co-eluting endogenous phospholipids The overall goal of this project is to evaluate the PK of cefazolin, ampicillin and sulbactam in patients undergoing surgery. To achieve this goal, one necessary step is to establish bioanalytical method(s) to quantify these antibiotics in human plasma. We developed a LC/MS/MS assay for quantification of cefazolin, ampicillin and sulbactam simultaneously. During assay development phase, the lower limit of quantification (i.e. LLOQ) of cefazolin was found to be 0.5 g/mL, with the signal to noise ratio being >50. However, during assay validation phase, both inter-day and intraday accuracy and precision of the LLOQ of cefazolin at 0.5 g/mL failed (i.e. out of the 20% acceptance criteria) in multiple runs, indicating that the signal was unstable. In addition, at the LLOQ level, the peak shape of cefazolin was poor, with peak shoulders shown in some but not all LLOQ calibration samples as well as some but not all analytical runs. We terminated the assay validation and went back
Tazobactam(g/mL) 0.25 0.5 1 5 10 50 100 150 – 0.25 0.75 75 120
Cefazolin(g/mL) 0.25 0.5 1 5 10 50 150 200 300 0.25 0.75 75 240
Sulbactam(g/mL) 0.5 1 5 10 20 50 100 150 200 0.5 1.5 75 180
Ampicillin(g/mL) 0.5 1 5 10 20 50 100 150 200 0.5 1.5 75 180
to assay development process. We suspected that the poor peak shape and unstable LLOQ of cefazolin may be caused by endogenous substances in matrix (i.e. human plasma). A simple protein precipitation method was used during the sample preparation process. This procedure can remove proteins but cannot efficiently eliminate lipids. Phospholipids are the main component of the cell membranes and glycerophosphocholines (PCs), including 2-lyso PC and diradyl PC, are the major class of phospholipids in the plasma (Fig. 1). To identify the impact of PCs on the elution and signal of cefazolin, we added a transition of m/z 184 > 184 to monitor PCs. The transition of 184 > 184 was used because both 2-lyso PC and diradyl PC are known to undergo in source collision-induced dissociation (CID) and generate fragment with same m/z ratio of 184, which allows for monitoring all PCs in one transition (i.e. 184 > 184). As shown in Fig. 2a, under the original LC gradient condition, cefazolin in a LLOQ calibration sample was eluted at 3.59 min with a clear shoulder on its peak. In addition, PCs were co-eluted at the same time with more than 100-fold higher peak intensity. To avoid the interference with PCs, a new LC gradient condition was used to separate cefazolin and PCs. As shown in Fig. 2b, under this new LC gradient condition, cefazolin and PCs were eluted at different time. As a result, peak shape, peak height, and peak intensity of cefazolin were all greatly improved. With this optimized condition, cefazolin LLOQ was reduced from 0.5 g/mL to 0.25 g/mL, assay
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Fig. 1. Structure of phospholipids. The glycerophosphocholines (PCs), the major class of phospholipids in the plasma, can be divided based on their different glycerin groups into 2-lyso PC (left) and diradyl PC (right).
validation was performed and all inter-day and intra-day accuracy and precision of cefazolin were successfully passed at the LLOQ level. As noted earlier, under the original assay condition, the peak shape of cefazolin was different among different analytical runs, and peak shoulder was observed in some but not all LLOQ samples. Since there are many different PCs, although they have same fragment and can be monitored together in one channel, they could be eluted at different times. We anticipate that the poor peak shape of cefazolin is due to the interference with different PCs, and the magnitude of interference may vary from sample to sample depending on the level of the different PCs. To test this hypothesis, blank human plasma from 6 different lots were processed using same protein precipitation method, and the elution patterns as well as intensity of PCs among these samples were compared. As shown in Fig. 3a, we found that multiple PC peaks were eluted with variable intensities. In our current assay, the protein precipitation method was performed using acetonitrile. Since methanol is also a common choice for protein precipitation, a natural question is that which solvent is more efficient in removing phospholipids in plasma. To address this question, a number of blank human plasma samples were precipitated with either acetonitrile or methanol and then the PCs in these samples were compared. As shown in Fig. 3, compared with samples extracted by acetonitrile (Fig. 3a), similar intensity of PCs were detected in samples extracted by methanol (Fig. 3b). Therefore, under the sample extraction procedure used in our study, acetonitrile is not more efficient in removing phospholipids in plasma than methanol. 3.2. [PTMC project, analyte effect] Ion suppression of stable-isotope-labeled internal standard due to co-eluting unlabeled analyte The overall goal of this project was to evaluate the PK of piperacillin, tazobactam, meropenem, and cefepime in patients from intensive care unit or patients with cystic fibrosis. We have successfully developed and fully validated a robust and sensitive LC/MS/MS assay for the quantification of these four antibiotics simultaneously. All the work was done under the GLP environment and we have reported the details of the assay validation result recently [6,7]. In this assay, 2 H5 -piperacillin was used as the IS for piperacillin and tazobactam, and 2 H6 -meropenem was the IS for meropenem and cefepime. Matrix/analyte effect was not observed during assay development or validation phases. However, this issue came up when clinical samples containing piperacillin and tazobactam were analyzed. Fig. 4 shows the typical chromatograms of piperacillin, tazobactam as well as 2 H5 -piperacillin in one calibration sample. Both piperacillin and tazobactam have same upper limit of quantification (i.e. ULOQ) of 150 g/mL. In one of the analytical runs, among 60 clinical samples analyzed, the cal-
culated concentrations of piperacillin in 17 samples were above the ULOQ and the calculated concentrations of tazobactam in all samples were within the ULOQ. The standard procedure is to accept tazobactam results, reject those piperacillin results outside of the calibration range, and rerun the samples after appropriate dilution for piperacillin quantification. However, after further data evaluation we noticed that the peak area of 2 H5 -piperacillin in those clinical samples with piperacillin higher than ULOQ was substantially lower than that in the calibration samples or clinical samples with piperacillin within ULOQ level (Fig. 5). The phenomenon was consistently observed in different batches which included those clinical samples with piperacillin concentrations higher than ULOQ. The most likely explanation for this phenomenon is ion suppression of 2 H5 -piperacillin when it was co-eluting with much higher concentration of piperacillin. Since 2 H5 -piperacillin was used as the IS of tazobactam, the abnormally low peak area of 2 H5 -piperacillin resulted in unreliable tazobactam quantification. The calculated tazobactam concentrations in those samples were much higher than the real concentrations. After careful examination, we rejected both piperacillin and tazobactam results obtained from clinical samples with piperacillin concentrations higher than ULOQ, and reran those samples with appropriate dilution. 4. Discussion In this paper, we presented two case examples on signal suppression caused by either endogenous phospholipids (matrix effect, example 1) or co-eluting analyte (analyte effect, example 2). Although the “syndrome” of these signal suppression is different, they are caused by the same fundamental mechanism and can be explained by Enke’s model for electrospray response [1]. During electrospray ionization process, droplets with a surface excess charge are created. As shown in Enke’s model, there is a limit on the number of excess charge sites available on the surface. As a result, competition for ionization can occur when more than one ionic species are eluted from the LC column at the same time and move to the ESI probe together [1]. It has been reported that under conditions commonly seen in LC/MS/MS for small-molecule compounds (e.g. flow rates > 0.1 mL/min; relatively high concentrations such as 10−6 to 10-4 M in the bulk phase), the total concentration of excess charge (Q) is typically close to 10-9 eq/L [8]. At such values of Q, the competition between analytes with abundant endogenous substances or other analytes becomes evident. During the process of competition for excess charge sites, between the two co-eluted ionic species, the one with much lower concentration will be affected more and consequently have more significant ion suppression. In case example 1, a matrix effect from co-eluting phospholipids was observed. The consequence of this matrix effect is the poor peak shape and unstable LLOQ of the co-eluting analyte (i.e. cefazolin) as well as the corresponding decreased assay sensitivity.
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Fig. 2. Representative LC/MS/MS chromatograms of phospholipids and cefazolin under a) old LC gradient condition and b) new LC gradient condition. The MRM transitions for phospholipids and cefazolin were m/z 184.0 > 184.0 and 453.3 > 320.7, respectively. LC gradient condition is expressed as % of acetonitrile.
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Fig. 3. LC/MS/MS chromatograms of phospholipids from samples prepared by protein precipitation method using a) acetonitrile or b) methanol. Samples were prepared from 6 different lots of human plasma with lithium heparin. LC gradient condition is expressed as % of acetonitrile.
Fig. 4. Representative LC/MS/MS chromatograms for piperacillin, tazobactam, as well as their internal standard 2 H5 -piperacillin. The MRM transitions for piperacillin, tazobactam, and 2 H5 -piperacillin were m/z 540.3 > 398.1, 301.4 > 168.1, and 545.3 > 398.1, respectively.
Our observation is consistent with literature reports. Phospholipids have been reported to interfere with many compounds and have been considered as the major cause of matrix effect [9,10]. Chambers et al. monitored the levels of various phospholipids in the samples to compare the relative cleanliness of final plasma extract from different sample preparation procedures [11]. They reported that protein precipitation is the least effective sample preparation method, while the mixed-mode strong cation exchange solid phase extraction (SPE) is the most effective method in eliminating phospholipids [11]. In addition, single step liquid-liquid extraction (LLE) with MTBE yields extracts of comparable cleanliness to mixed mode cation SPE [11]. To overcome the phospholipids-mediated ion suppression, there are following two main strategies: 1) To improve the cleanliness of final plasma extract by switching protein precipitation method to more efficient LLE or SPE. However, as both LLE and SPE are time consuming and labor intensive, this strategy may not be suitable for the situation where high throughput sample analysis is required. Although protein precipitation does not provide very clean final extract, its advantage of simple, fast and less labor intensive allows for high throughput sample analysis. In our projects, high throughput sample analysis is required as thousands
Fig. 5. Peak area of piperacillin (circles) and 2 H5 -piperacillin (triangles) in calibration standards as well as 17 clinical samples. Final concentration of 2 H5 -piperacillin spiked in each sample was 2.5 g/mL. Although same concentration of 2 H5 -piperacillin was spiked in each sample, the peak area of 2 H5 -piperacillin in those 17 piperacillin clinical samples (right panel) was substantially lower than that in piperacillin calibration standard samples (left panel). For those 17 clinical samples, the concentrations of piperacillin were all higher than the ULOQ (i.e. 150 g/mL).
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of clinical samples need to be analyzed. In this case, the strategy of switching protein precipitation method to LLE or SPE is not preferred. 2) To optimize the LC condition to separate the analyte peak and phospholipids peaks. This is the strategy we preferred and used in our project since it does not conflict with high throughput sample analysis. Considering that phospholipids represent the major source of matrix effect, adding the transition of 184 > 184 to monitor PCs will be highly valuable and should be considered during assay development phase. In addition, using different ionization method such as APCI may also avoid significant matrix effect since APCI is known to be less prone to ion suppression than ESI. In case example 2, an analyte effect on stable-isotope labeled analyte was observed and it was caused by the co-eluting unlabeled analyte. In the current example, this analyte effect was manifested as a significant decrease of the internal standard (2 H5 -piperacillin) peak area when the concentration of the unlabeled analyte (i.e. piperacillin) in clinical samples exceeded a threshold. It should be noted that this analyte effect on 2 H5 -piperacillin may not have significant impact on the calculated concentration of piperacillin as the ratio of piperacillin and 2 H5 -piperacillin signals should still be relatively proportional to the ratio of piperacillin’s concentrations. However, this analyte effect can become a serious problem for multi-analyte procedures in which the stable-isotope labeled internal standard is not only used as internal standard for the respective unlabeled analogue, but also for other analytes, which is the exact situation that we encountered. As piperacillin-mediated ion suppression on 2 H5 -piperacillin did not occur in calibration standard samples and only happened in those clinical samples containing piperacillin higher than 150 g/mL, this analyte effect can be easily overlooked and consequently substantial quantification bias on tazobactam would result. Therefore, for such multi-analyte procedures, it is important to carefully examine the peak area of the stable-isotope-labeled internal standard before accepting the calculated concentrations of the other analytes. In case study 2, we showed an example of ion suppression on internal standard by the co-eluting high concentration of analyte. Theoretically, there could be a mutual interaction between the analyte and co-eluting internal standard. This means that if the concentration of the internal standard is high, it potentially could cause the ion suppression of the co-eluting analyte. This mutual suppression between analyte and co-eluting internal standard has been reported by Sojo et al., and they commented that co-eluting internal standard could have substantial impact on the method detection limits [8]. In their study, the IS concentrationdependent ion suppression clearly shown, indicating that selection of the concentration of the internal standard is of importance [8]. Traditionally, the concentration of the internal standards is usually selected to be midway of the calibration range. This approach may cause significant ion suppression of the analyte in those calibration standard samples containing low concentrations of analyte, resulting in increase in detection limit (i.e. decreased sensitivity). Different strategies may be needed depending on the type of internal standard. If the internal standard is a different compound compared with the analyte, mobile phase condition should be optimized to ensure that IS and the analyte are not eluted at the same time. If peak separation is not successful, different internal standard should be considered. If the internal standard used in the method is stable-isotope-labeled analyte, peak separation between IS (labelled analyte) and analyte itself is impossible. In this case, the concentration of labeled IS must be selected carefully with the goal of maintaining the desired detection limit of the analyte. The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: one compromise is made between sample cleanliness
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and sample preparation speed since it is known that protein precipitation does not provide very clean final extract [12]; another compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has be to accepted. It should keep in mind that matrix effect may occur when the final sample extract is not clean and analyte effect may occur when there is overlap or co-elution of some peaks. Therefore, during assay development process, it is necessary to add a channel to monitor phospholipids and make sure that they are not eluted at the same time with the analyte(s). During assay validation process, it is important to evaluate not only matrix effect but also the interference between analytes and ISs. The evaluation of the interference between analytes and ISs is particularly important when they are co-eluted, such as isotope-labeled IS where peak separation is not possible. As noted earlier, the concentration of labeled IS must be selected carefully to make sure that the LLOQ of the analtye is not significantly affected. Similarly, the effect of analyte at the ULOQ level on the co-eluting IS should also be evaluated to make sure that the peak area of the IS is not significantly reduced. Author statement Guohua An: Conceptualization, methodology, Writing- Original draft preparation, Funding acquisition Thanh Bach: methodology, experiment and data collection, Manuscript review and editing Inas Abdallah: experiment and data collection Demet Nalbant: experiment and data collection Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Partial support was provided by the Division of Microbiology and Infectious Disease, the National Institute of Allergy and Infectious Disease, the National Institutes of Health (HHSN272200800008C) References [1] C.G. Enke, A predictive model for matrix and analyte effects in electrospray ionization of singly-charged ionic analytes, Anal. Chem. 69 (23) (1997) 4885–4893. [2] T.M. Annesley, Ion suppression in mass spectrometry, Clin. Chem. 49 (7) (2003) 1041–1044. [3] F. Gosetti, E. Mazzucco, D. Zampieri, M.C. Gennaro, Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry, J. Chromatogr. A 1217 (25) (2010) 3929–3937. [4] F.T. Peters, D. Remane, Aspects of matrix effects in applications of liquid chromatography-mass spectrometry to forensic and clinical toxicology–a review, Anal. Bioanal. Chem. 403 (8) (2012) 2155–2172. [5] C. Ghosh, C.P. Shinde, B.S. Chakraborty, Influence of ionization source design on matrix effects during LC-ESI-MS/MS analysis, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 893-894 (2012) 193–200. [6] R. D’Cunha, T. Bach, B.A. Young, P. Li, D. Nalbant, J. Zhang, P. Winokur, G. An, Quantification of Cefepime, Meropenem, Piperacillin, and tazobactam in human plasma using a sensitive and robust liquid chromatography-tandem mass spectrometry method, part 1: assay development and validation, Antimicrob. Agents Chemother. 62 (9) (2018). [7] R. D’Cunha, T. Bach, B.A. Young, P. Li, D. Nalbant, J. Zhang, P. Winokur, G. An, Quantification of Cefepime, Meropenem, Piperacillin, and tazobactam in human plasma using a sensitive and robust liquid chromatography-tandem mass spectrometry method, part 2: stability evaluation, Antimicrob. Agents Chemother. 62 (9) (2018). [8] L.E. Sojo, G. Lum, P. Chee, Internal standard signal suppression by co-eluting analyte in isotope dilution LC-ESI-MS, Analyst 128 (1) (2003) 51–54. [9] O.A. Ismaiel, M.S. Halquist, M.Y. Elmamly, A. Shalaby, H.T. Karnes, Monitoring phospholipids for assessment of matrix effects in a liquid
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[11] E. Chambers, D.M. Wagrowski-Diehl, Z. Lu, J.R. Mazzeo, Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 852 (1-2) (2007) 22–34. [12] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle, The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds, Rapid Commun. Mass Spectrom. 13 (12) (1999) 1175–1185.