A validated LC–MS/MS method for the quantification of piperacillin/tazobactam on dried blood spot

2795Bioanalysis (2014) 6(21), 2795–2802 ISSN 1757-6180

part of

MethodologySpecial Focus Issue: Clinical Chemistry

10.4155/BIO.14.205 © 2014 Future Science Ltd

Bioanalysis

10.4155/BIO.14.205

Methodology

Barco, Risso, Bruschettini et al.A validated LC–MS/MSmethod for the quan-

tification of piperacillin/tazobactam on DBS

6

21

2014

Background Therapeutic drug monitoring (TDM) of antibiotics in children is of fundamental importance for effective patient management. The use of dried blood spot (DBS) offers a number of advantages over conventional blood collection. The aim of this study is to develop and validate a method to measure piperacillin/tazobactam in DBS. Results The analysis was performed by using LC–MS/MS operating in multiple reaction monitoring mode. The method has been validated by applying EMA guidelines and its suitability for TDM was evaluated by using samples from low birth weight neonates. Conclusion This paper describes a fast and cost-effective micromethod for the simultaneous determination of piperacillin/tazobactam levels on dried blood spot that is suitable for TDM in children.

BackgroundThe use of dried blood spot (DBS) sam-pling in combination with LC–MS/MS for the quantitative analysis of drugs has an emerg-ing role in the clinical pathology laboratory, especially in pediatrics. DBS offers a number of advantages over conventional sample col-lection [1–4]. The smaller sample volume and the less invasive sampling method (heel prick rather than conventional venous cannula) make it an attractive procedure also for low birth weight (LBW) neonates. Furthermore, DBS usually offers a simpler storage and easier transfer being most molecules stable in DBS at room temperature [5]. DBS are routinely used for neonatal screening of metabolic disorders, cystic fibrosis and hypothyroidism [6]. The use of DBS in combination with LC–MS/MS has been increasingly employed in clini-cal studies and TDM of various molecules including immunosuppressants [7], antipyretic [8], antimalarial [9], anticonvulsivants [10] and antiretrovirals [11]. DBS-LC–MS/MS has been demonstrated to be a reliable and valid technique also for antibiotics [12]. Therapeutic drug monitoring (TDM) is of fundamental importance in individualizing antimicrobial therapy, in particular for LBW neonates but the scarce data on the PK in this population

limit the routine use of TDM for antibiotics [13,14]. The availability of reliable and validated bioanalytical methods for the determination of these molecules in limited sample volumes is of crucial importance for the conduct of PK studies and consequent TDM. In the pres-ent work we describe a simple and fast DBS- LC–MS/MS method for the simultaneous determination of piperacillin/tazobactam. Piperacillin/tazobactam is a combination antibiotic containing the extended-spectrum penicillin antibiotic piperacillin and the β-lactamase inhibitor tazobactam. The com-bination has activity against many Gram-positive and Gram-negative pathogens and anaerobes, including Pseudomonas aeruginosa [15] and it is employed for the management of pediatric patients with severe infection com-plications. The performance and suitability of the assay have been evaluated by applying an extensive validation protocol based on EMA guidelines [16], and by analyzing DBS samples from pediatric patients undergoing antibiotic treatment.

ExperimentalChemicals & reagentsPiperacillin, tazobactam, and prazosin (IS) were purchased from Sigma Aldrich (Milan,

A validated LC–MS/MS method for the quantification of piperacillin/tazobactam on dried blood spot

Sebastiano Barco1, Francesco Maria Risso2, Matteo Bruschettini2, Roberto Bandettini1, Luca A Ramenghi2, Gino Tripodi1, Elio Castagnola3 & Giuliana Cangemi*,1

1Clinical Pathology Laboratory Unit,

Istituto Giannina Gaslini, Genoa, Italy 2Neonatal Intensive Care Unit, Istituto

Giannina Gaslini, Genoa, Italy 3Infectious Disease Unit, Istituto Giannina

Gaslini, Genoa, Italy

*Author for correspondence:

Tel.: +39 0 105636835

Fax: +39 0 103994168

[email protected]

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Methodology Barco, Risso, Bruschettini et al.

Italy). Water was purified by reverse osmosis and fil-trated through a Milli-Q purification system (Mil-lipore, Milford, MA, USA). HPLC grade methanol was purchased from Sigma Aldrich Srl (Milan, Italy) and formic acid (99.9%) from Merck (Darmstadt, Germany).

Preparation of calibrators & QC samplesStock solutions (2 mg/ml for piperacillin and 400 μg/ml for tazobactam) were prepared by dissolving each pow-dered antibiotic in water. In order to set up and validate the method whole blood from healthy adult donors was spiked with piperacillin and tazobactam at differ-ent concentrations. Two different working solutions were obtained by diluting the stock solutions 1:10 with whole blood (200 μl of each stock solution was added to 1,8 ml whole blood to obtain 200 μg/ml and 40 μg/ml working solution of piperacillin and tazobac-tam respectively). Calibrators and QCs were obtained by serial dilution with whole blood. A 20-μl volume of each fortified blood was spotted on filter paper and used as calibrators or QC. Two different six-point cali-bration curves were created to yield concentrations of: 0.6–1.2–2.5–5–10–20 and 100 μg/ml for piperacillin and 0.1–0.4–1–4–10 and 40 μg/ml for tazobactam, respectively. QC samples were prepared at the follow-ing four concentration levels: 0.6 μg/ml (LLOQ), 1.5 μg/ml (low QC), 50 μg/ml (medium QC) and 75 μg/ml (high QC) for piperacillin and 0.1 μg/ml (LLOQ), 0.25 μg/ml (low QC), 20 μg/ml (medium QC) and 30 μg/ml (high QC), for tazobactam.

Extraction from DBSEach DBS sample was punched obtaining a 3.2 mm diameter disk (containing about 3 μl of blood) into a vial and extracted with 100 μl methanol/water 70:30 (V/V) solution containing 220 μg/ml IS. The extrac-tion was performed using an orbital shaker for 60 min at 25°C and then the solution was transferred into a new vial and analyzed by LC–MS/MS.

Human samplesLeftover EDTA whole blood samples from rou-tine hemocromocytometric tests from healthy adult

volunteers have been used for the development and validation of the present method. Samples were obtained from preterm infants of the Neonatal Intensive Care Unit of Istituto Giannina Gaslini, Genoa-Italy with suspected systemic infections who received intravenous piperacillin/tazobactam (50–100 mg/Kg q 8 h) based on gestational and postnatal age. Sparse samples were obtained after first and multiple doses. DBS was pre-pared by spotting whole blood onto filter paper cards (Ahlstrom 226, LTA srl, Milan, Italy), let to dry and stored at 4°C in a sealed plastic bag containing desic-cant until analysis. Parents’/guardian written informed consent were given before any study-related procedure.

Chromatographic conditionsGradient separation chromatography was carried out using a Kinetex C18 column (100 mm × 4.6 mm, i.d. 2.6 μm, Phenomenex, Bologna, Italy) with mobile phase A consisting of 0.1% formic acid in water and mobile phase B of acetonitrile. The starting composition of the mobile phase was 95% phase A. The percentage of sol-vent B was programmed to reach 100% in 3 min at a flow rate of 700μl/min, then the column was washed with 100% B for 1 min and finally reconditioned at 95% A for 2 min for a total run time of 5.5 min. The column temperature was maintained at 40°C and injec-tion volume was 10 μl. At these chromatographic con-ditions, piperacillin, tazobactam and IS have retention times of 3.43, 2.71 and 3.03 min, respectively.

Mass spectrometric conditionsMass spectrometric detection was performed using a Thermo Scientific TSQ Quantum Access MAX triple quadrupole system. Ionization was achieved using elec-trospray in the positive ion mode for piperacillin and IS and in the negative ion mode for tazobactam. The spray voltage was set at 3500 V in positive polarity and 3000 V in negative polarity. Nitrogen was used as the nebu-lizer and auxiliary gas, set at 50 and 15 arbitrary units, respectively. Vaporizer and capillary temperature set-tings were 350 and 275°C. For collision-induced disso-ciation, high purity argon was used at a pressure of 1.5 mTorr. Piperacillin and IS were detected using multiple reaction monitoring (MRM) of the following specific transitions: 540→397.7 and 384→246.8 respectively. The parent ion used for piperacillin (MW 517.55) is consistent with the (+23 da) sodium adduct [M+Na]+ of piperacillin. Tazobactam was detected using MRM of the following transitions: 298.9→138.1, 254.9.

Method validationAn improved laboratory scheme based on EMA guide-line on bioanalytical method validation [16,17] was used for assay validation.

Key terms

Dried blood spot: A form of biosample in which blood samples are blotted and dried on filter paper

LC–MS/MS: Analytical technique which allows very specific and sensitive quantification of drugs.

Piperacillin: Extended spectrum beta-lactam antibiotic. It is normally used together with a beta-lactamase inhibitor, notably in the combination piperacillin/tazobactam.

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A validated LC–MS/MSmethod for the quantification of piperacillin/tazobactam on DBS Methodology

SelectivitySelectivity was evaluated by analyzing six different batches of whole blood. For each batch, three differ-ent samples were analyzed: a blank sample, a sample spiked with piperacillin and tazobactam at the LLOQ and a third sample spiked with IS. The absence of interfering components was accepted when the signal was less than 20% of the LLOQ for the analytes and less than 5% for the IS.

Carry- overCarry-over was assessed by injecting blank samples in triplicate after the highest calibration standard. The signal in the blank sample following the high standard should not be greater than 20% of the LLOQ and 5% for the IS.

Matrix effects & extraction recoveriesMatrix effects and extraction recoveries of piperacillin and tazobactam were assessed at two different levels (corresponding to the low and high QC) analyzed in triplicate. Matrix effects were assessed by measuring the ionization recoveries obtained by comparing peak area of the analytes spiked after extraction from DBS to peak area of pure solutions at the same concentra-tion [18]. Extraction recoveries were determined by comparing peak area of analytes spiked before extrac-tion to peak area of analytes spiked after extraction. Global recovery was obtained by comparing peak area of analytes spiked before extraction to peak area of pure solutions at the same concentrations.

LinearityLinearity was evaluated by analyzing calibration stan-dards three times on three separate days in the test range. The slope, intercept, and correlation coefficient for each calibration curve were estimated by plotting the peak area ratio of analyte IS vs. the analyte con-centration of each calibration standard sample using a 1/x weighting factor. The acceptance criteria for the amounts of back-calculated standard was ± 15% of the theoretical value (except ± 20% for the lowest standard)

Accuracy & precisionWithin-run and between-run precision and accuracy were determined at four concentration levels, using the four level QC samples (LLOQ, low, medium and high QC) with each sample being analyzed six sepa-rate times. Inter-assay imprecision was estimated by repeating the test six times on three separate days. Accuracy was expressed as the mean relative error (i.e., the agreement between test results and the expected value expressed as percentage). Precision was expressed as the coefficient of variation (CV%). The acceptance criteria for within- and between-run precision was ≤15% and for accuracy was 85–115% of the nominal concentrations.

LLOQThe LLOQ was determined as the sample concentra-tion that provided measurements with an imprecision ≤20% and accuracy within 80–120% of the nominal concentration.

StabilityShort term and long term stability of QCs were deter-mined from three replicate assays of low and high QC after maintaining DBS at 25°C for one week and at -20°C for one month respectively. Stability of extract samples was determined after maintaining extracts at 25°C for over 6 h. Freeze-thaw sample stability was determined after three freeze and thaw cycles of DBS. Stock solution stability was evaluated after keeping it at room temperature for 6 h.

Results & discussionIn this paper we describe a fast, reliable and cost effective assay for the quantification of piperacillin/tazobactam from DBS. The availabil-ity of reliable and validated analytical methods for the quantification of these drugs is of fundamental importance. The method that we have developed allows the simultaneous identification of piperacil-lin and tazobactam from a low volume of blood. The method has been fully validated according to interna-tional guidelines.

Table 1. Results of the ionization, extraction and global recoveries obtained for two quality control levels.

QC level Ionization recoveries Extraction recoveries Global recoveries

Piperacillin QC I 96.5 11.8 11.4

QC II 94.5 11.2 11.3

Tazobactam QC I 105.2 3.6 3.8

QC II 98.2 3.7 3.5

QC: Quality control.

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Methodology Barco, Risso, Bruschettini et al.

Method developmentSample preparation was optimized taking into account the following variables: impact of blood volume spotted, impact of spotting with capillary or pipette, extraction solution composition, volume, time and temperature. The best diffusion in the fil-ter paper was achieved by spotting a 20 μl volume of blood. The use of capillary or pipette gave simi-lar results. Different extraction mixtures containing methanol or acetonitrile mixed with water at differ-ent percentages (from 30% to 70%) were compared. The most efficient extraction solution with the high-est extraction yield was found to be methanol/water 70:30 (V/V). The volume of extraction solution was optimized in order to minimize sample dilution. The extraction time was also optimized in order to obtain the fastest procedure without loss of analyte. A 60 min extraction was obtained. Different extrac-tion temperatures (25°C, 37°C and 60°C) were also tested. The best extraction yield occurred at 25°C. The method that we have developed is based on a non-deuterated IS. The use of stable isotope-labeled internal standards may be more appropriated in order to achieve accurate quantization of analytes in

complex matrices. Nevertheless labeled standards, when available, are very expensive and not appropri-ate for the development of cost-effective laboratory methods.

Method validationSelectivityMethod selectivity was established by demonstrating the absence of interference at the specific ion transi-tions that were selected for analyte quantification by using six different batches of blank samples. For each blank sample, signal was below 20% of the LLOQ for piperacillin and tazobactam and 5% for IS, ensuring the selectivity of the method.

Carry-overInjection of blank samples in triplicate directly after the highest calibration standard showed a signal cor-responding to 0.6 and 0.07% of the LLOQ at the first injection, to 0.1 and 0.02% at the second injection for piperacillin and tazobactam respectively, whereas a signal below 0.05% of the working concentration was observed for IS. Despite this slight carry-over on piperacillin, acceptance criteria were fulfilled.

Table 2. Within- and between-run precision and accuracy.

Total Amount found (mean ± S.D.) (μg/ml)

CV (%) Accuracy (%)

Inter-assay

Piperacillin

7.5 7.4±0.5 6.1 98.1

15 14.8±1.7 11.7 99.0

30 33.3±2.1 6.3 111.1

60 63.1±5.9 9.3 105.2

Tazobactam (μg/ml)

1.5 1.6±0.1 8.6 105.4

3 3.41±0.3 9.7 112.3

6 6.6±0.6 9.6 110.0

12 13.0±1.4 11.1 108.4

Piperacillin (μg/ml)

7.5 6.9±0.7 10.6 92.1112

15 16.8±1.1 6.5 112.1

30 28.0±1.9 6.7 93.3

60 64.1±5.7 9.0 106.8

Tazobactam (μg/ml)

1.5 1.5±0.1 9.3 103.3

3 3.2±0.1 2.6 106.1

6 6.7±0.5 7.5 111.0

12 12.5±1.1 8.9 104.3

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A validated LC–MS/MSmethod for the quantification of piperacillin/tazobactam on DBS Methodology

Matrix effects &extraction recoveriesResults of the determination of ionization, extrac-tion and global recoveries obtained for 2 QC levels are summarized in Table 1. The ionization recover-ies obtained were high for both analytes. On the contrary, very low extraction recoveries could be obtained even in the optimized conditions. The

results could be considered acceptable as they are highly reproducible.

LinearityThe linear regression fit for the calibration curves was achieved for both analytes with R2>0.9978. All back-calculated values did not differ from ± 15% of the

Figure 1. Chromatograms obtained for piperacillin, tazobactam and internal standard at the lower limit of quantification. AA: Peak area; NL: Normalized level; RT: Retention time; SN: Signal to noise ratio.

100

NL: 1.19E3RT: 2.71AA: 2760SN: 113

Piperacillin

Prazosin

Tazobactam

80

60

40

20

0

100NL: 3.87E5

Rel

ativ

e ab

ou

nd

ance

RT: 3.03AA: 832415SN: 6045

80

60

40

20

0

100NL: 5.56E4

Time (min)

RT: 3.43AA: 124785SN: 1297

80

60

40

20

02.4 2.6 2.8 3.0 3.2 3.4

N

S

HOO

O

NH

NH

N

O

O

H

NO

O

H3C

CH3

CH3

NN N

N

S

O OH

O

H O O

CH3

N

NN

N

O

O

OO

CH3

CH3

NH2

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Methodology Barco, Risso, Bruschettini et al.

theoretical value. The mean equations obtained were y= 0.4+0.14× and y= -0.001+0.02× for piperacillin and tazobactam respectively.

Accuracy & precisionWithin- and between-run precision and accuracy were all within the established ranges of acceptance as summarized in Table 2.

LLOQThe LLOQ obtained for piperacillin and tazobactam were 0.6 and 0.1 μg/ml with precision and accuracy of 13.4 and 18.7% respectively. Figure 1 shows the chro-matograms obtained from the two antibiotics at the LLOQ.

StabilityThe results of the stability tests are shown in Table 3. Piperacillin and tazobactam resulted to be stable in DBS for prolonged storage at -20°C, while a rapid degradation occurred at 25°C. Usually DBS sampling procedure helps the molecule stabilization allowing prolonged storage at room temperature thus facilitat-ing the shipment and delivery of samples. In this case attention must be paid to shipment and storage of DBS that must be performed at -20°C. The processed sam-ple resulted to be stable at least for 6 h at 25°C thus ensuring no degradation on autosampler.

Application to clinical samplesThe present method has been applied to quantify piper-acillin/tazobactam in DBS obtained from LBW neonates

under treatment with the analyzed antibiotics. Seven samples were obtained from four patients (two males and two females, 16 days mean postnatal age, 30 weeks mean gestational age. As expected the antibiotic levels measured were in a wide range of concentrations (4.5–68.9 and 0.2–7.8 μg/ml for piperacillin and tazobactam respec-tively). The samples quantified gave concentrations, which are within the calibration curve and the choice of QC samples are correct for these levels. Further studies are needed to compare piperacillin/tazobactam concen-trations in DBS and plasma taking in consideration an hematocrit correction for DBS.

ConclusionIn this paper we describe a fast and cost-effec-tive assay for the simultaneous determination of piperacillin/tazobactam concentrations in DBS. The method has been fully validated according to EMA guidelines. Its robustness has been tested both on QC and patient samples. The simple extraction procedure and LC–MS/MS protocol result in a very short turn-around time, making it an attractive procedure for PK studies.

Future perspectiveThe present method is compatible with the sample vol-ume limitations required in pediatric because it uses a very small sample volume. We believe that the described LC–MS/MS method is suitable for application in chil-dren and neonates for PK studies and consequent rou-tine TDM using DBS as a tool to optimize antibiotic therapy in seriously ill children.

Table 3. Results of the stability tests.

Storage Condition Concentration (μg/ml) Recovery (%) CV (%) †

Piperacillin Tazobactam Piperacillin Tazobactam Piperacillin Tazobactam

Stock solution 6 h at +25°C

2000 500 92 98 4 5

DBS sample one week at +25°C

15 3 56 30 7 8

60 12 50 24 3 10

Extracted sample 6 h at +25°C

15 3 95 98 5 6

60 12 91 95 6 4

DBS 3 freeze–thaw cycles at -20°C

15 3 95 101 2 4

60 12 93 98 6 4

DBS sample one month at -20°C

15 3 101 93 9 6

60 12 95 90 4 5†CV calculated from three replicatesDBS: Dried blood spots.

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ReferencesPapers of special note have been highlighted as:

• of interest; •• of considerable interest

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6 La Marca G. Mass spectrometry in clinical chemistry: the case of newborn screening. Pharm. Biomed. Anal. 28, (2014).[Epub ahead of print].

• DescribesthestateoftheartoftheuseofMSfornewbornscreening.

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monitoring using limited sampling strategy and abbreviated AUC estimation. Transpl. Int. 21, 140–145 (2008).

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A validated LC–MS/MSmethod for the quantification of piperacillin/tazobactam on DBS Methodology

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-

ment with any organization or entity with a financial inter-

est in or financial conflict with the subject matter or mate-

rials discussed in the manuscript. This includes employment,

consultancies, honoraria, stock ownership or options, expert

testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this

manuscript.

Ethical conduct of researchThe authors state that they have obtained appropriate in-

stitutional review board approval or have followed the prin-

ciples outlined in the Declaration of Helsinki for all human

experimental investigations. In addition, for investigations

involving human subjects, informed consent has been ob-

tained from the participants involved or from patients’

parents/guardians.

Executive summary

Background• The use of DBS-LC–MS/MS for the quantitative analysis of drugs has an emerging role in the clinical pathology

laboratory• The availability of reliable and validated bioanalytical methods for the determination of antibiotics in limited

sample volumes is of crucial importance for the conduct of PK studies and consequent therapeutic drug monitoring (TDM) in neonates.

Experimental• A DBS-LC–MS/MS method for the simultaneous quantification of piperacillin and tazobactam has been

developed• The method has been extensively validated by applying EMA guidelines on bioanalytical method validationResults• The method is fast and simple and shows excellent performance on quality control (QC) samples.• Attention must be paid to shipment and storage of dried blood spot (DBS) that must be performed at -20°C.• The performance of the method has been confirmed on clinical samples derived from children under

treatment with the analyzed antibiotics

2802 Bioanalysis (2014) 6(21)

14 Cohen-Wolkowiez M, Watt KM, Zhou C et al. Developmental pharmacokinetics of piperacillin and tazobactam using plasma and dried blood spots from infants. Antimicrob. Agents Chemother. 58, 2856–2865 (2014).

15 Sturm AW, Allen N, Rafferty KD et al. Pharmacokinetic analysis of piperacillin administered with tazobactam in critically ill, morbidly obese surgical patients. Pharmacotherapy. 34, 28–35 (2014).

16 EMEA/CHMP/EWP/192217/2009. Guideline on bioanalytical method validation. The European Medicines Agency. www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08503/WC500109686.pdf

17 Cangemi G, Barco S, Verrina EE et al. Micromethod for quantification of cinacalcet in human plasma by liquid chromatography-tandem mass spectrometry using a stable isotope-labeled internal standard. Ther. Drug Monit. 35, 112–117 (2013).

18 Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS.MS. Anal. Chem. 75, 3019–3030 (2003).

•• Describesoneofthemostwidelyusedmethodformatrixeffectassessment.

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