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This article was downloaded by: [University of Kent]On: 09 November 2014, At: 09:03Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
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DEVELOPMENT AND VALIDATIONOF A HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY METHODTO DETERMINE VANCOMYCINCONCENTRATIONS IN PLASMA AND PIGPULMONARY TISSUELaura Guerrero a , Pilar Martínez-Olondris b , Montserrat Rigol c ,Mariano Esperatti b , Néstor Luque b , Antoni Torres b & Dolors Soy aa Pharmacy Service, Hospital Clínic Barcelona, Institutd'Investigacions Biomèdiques Agustí Pi i Sunyer (IDIBAPS), Universityof Barcelona , Barcelona , Spainb Pneumology Service, Institut Clínic del Tòrax, Hospital ClínicBarcelona, Institut d'Investigacions Biomèdiques Agustí Pi i Sunyer(IDIBAPS), Universidad de Barcelona , Barcelona , Spainc Cardiology Service, Institut Clínic del Tòrax, Hospital ClínicBarcelona, Institut d'Investigacions Biomèdiques Agustí Pi i Sunyer(IDIBAPS), Universidad de Barcelona , Barcelona , SpainPublished online: 30 Jan 2012.
To cite this article: Laura Guerrero , Pilar Martínez-Olondris , Montserrat Rigol , Mariano Esperatti ,Néstor Luque , Antoni Torres & Dolors Soy (2012) DEVELOPMENT AND VALIDATION OF A HIGHPERFORMANCE LIQUID CHROMATOGRAPHY METHOD TO DETERMINE VANCOMYCIN CONCENTRATIONS INPLASMA AND PIG PULMONARY TISSUE, Journal of Liquid Chromatography & Related Technologies, 35:2,240-257, DOI: 10.1080/10826076.2011.597073
To link to this article: http://dx.doi.org/10.1080/10826076.2011.597073
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DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCELIQUID CHROMATOGRAPHY METHOD TO DETERMINEVANCOMYCIN CONCENTRATIONS IN PLASMA AND PIGPULMONARY TISSUE
Laura Guerrero,1 Pilar Martınez-Olondris,2 Montserrat Rigol,3
Mariano Esperatti,2 Nestor Luque,2 Antoni Torres,2 andDolors Soy1
1Pharmacy Service, Hospital Clınic Barcelona, Institut d’Investigacions BiomediquesAgustı Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain2Pneumology Service, Institut Clınic del Torax, Hospital Clınic Barcelona, Institutd’Investigacions Biomediques Agustı Pi i Sunyer (IDIBAPS), Universidad de Barcelona,Barcelona, Spain3Cardiology Service, Institut Clınic del Torax, Hospital Clınic Barcelona, Institutd’Investigacions Biomediques Agustı Pi i Sunyer (IDIBAPS), Universidad de Barcelona,Barcelona, Spain
& Simple and specific analytical methods were developed and validated to quantify vancomycinin plasma and lung tissue, which were obtained from a model of pneumonia in mechanicallyventilated piglets. Tissue and plasma samples were precipitated and centrifuged; 100 lL of thesupernatant were injected into the chromatographic system. Ceftazidime was used as the internalstandard. The stationary phase was a silica based column Symmetry300 C18 (150� 4.6mm) withpre-column. The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 75mM sodiumacetate buffer (pH¼ 3) with (B) acetonitrile (92%=8%;v=v). Isocratic flow rate was set at 0.8mL=min and 0.7mL=min for plasma and tissue samples, respectively. UV absorbance detection was setat 230 nm. Standard curves showed good linearity for plasma and pulmonary tissue (r2> 0.99).The lower limits of quantitation were 1.56 lg=mL and 3.13 lg=mL for plasma and tissue, respect-ively. The intra- and inter-day precisions and accuracy all satisfied the acceptance criteria. BothHPLC assays to quantify vancomycin in plasma and pulmonary tissue are rapid, simple, andinexpensive. These methods could be helpful to develop further pharmacokinetic studies ofvancomycin penetration in pulmonary tissue.
Keywords animal model, high performance liquid chromatography, lung tissue,pharmacokinetics, plasma, vancomycin
Address correspondence to Dolors Soy, PharmD, PhD, Pharmacy Service, Hospital ClınicBarcelona, C=Villarroel, 170-08036, Barcelona, Spain. E-mail: [email protected]
Journal of Liquid Chromatography & Related Technologies, 35:240–257, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.597073
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INTRODUCTION
Vancomycin is a glycopeptide antibiotic used in the treatment of seriousgram-positive infections, mainly due to penicillin-resistant staphylococciand enterococci, or Meticillin-resistant Staphylococcus aureus (MRSA).However, vancomycin distribution to deep tissues, such as pulmonarysegments,[1] cardiac vegetations,[2] or osteoarticular material.[3] is quitevariable and, thus, microbiological failure could be expected.
High performance liquid chromatography (HPLC) can be consideredan adequate technique for determining vancomycin in plasma and pul-monary tissue. The sensitivity and precision of HPLC and its applicabilityto a wide variety of compounds has led to its use in clinical laboratoriesfor monitoring of a variety of therapeutic agents.[4] A variety of methodshave been developed to analyze vancomycin in biological samples. Someof these techniques used solid-phase extraction (SPE) cartridges to separ-ate vancomycin from the original matrix.[5–8] Others precipitated withmethanol or acetonitrile.[9,10] Favetta et al.[11] used an electrochemicaldetection to quantify vancomycin in plasma.
The aim of this study is to develop simple and reliable high-performance liquid chromatographic (HPLC) assays to quantify vancomy-cin in plasma and lung tissue obtained from a model of pneumonia inmechanically ventilated piglets.
EXPERIMENTAL
Chemicals and Reagents
Lyophilized vancomycin and Ceftazidime, the internal standard (IS),were obtained from NORMON, S.A. (Madrid, Spain) and IPS Farma, S.L.(Madrid, Spain), respectively. Perchloric acid 70% PRS and Sodium acetate3-hydrate, �98% were purchased from Panreac (Barcelona, Spain) andFagron Iberica. S.A.U. (Terrassa, Spain). Acetonitrile gradient 240=far-UV-HPLC grade was acquired from Sharlau, S.A. (Barcelona, Spain) and the10xTBE Buffer Ultrapure (1M Tris, 0.9M Boric Acid, 0.01M EDTA) fromInvitrogen (Paisley, Scotland).
Instrumentation
HPLC was performed using an Agilent 1100 liquid chromatographysystem (Agilent technologies Spain, S.L., Madrid, Spain) consisting of avacuum degasser, a gradient pump, an auto sampler, and a variable wave-length ultraviolet (UV) detector. Data acquisition was performed using theappropriate software (Agilent Chemstation Rev.B.03.01) for signal treatment.
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High-Performance Liquid Chromatography (HPLC)
HPLC techniques used in this study are based on the methods pro-posed by Lopez et al.[10] and Ye et al.[12] to analyze vancomycin in plasma.
In the present study, for both methods, the stationary phase was a silicabased column Symmetry300C18 (150� 4.6mm) with pre-column fromWaters Corporation, (Milford, MA, USA). The mobile phase consisted of20% ultrafiltered water and 80% of (A) 75mM sodium acetate buffer(pH¼ 3) with (B) acetonitrile (92%=8%; v=v). The flow rate was fixed at0.8mL=min and 0.7mL=min for plasma and tissue samples, respectively,and the injection volume was set to 100 mL. UV absorbance detection waspredetermined at 230 nm.
Drug Analysis and Calibration in Plasma
Calibration curves were prepared in free-drug plasma. The observedpeak-area ratio method with reference to the internal standard was appliedto assess drug concentration.
Calculation of the unknown vancomycin concentration in humanplasma samples was performed using the linear regression equation ofthe peak area ratio against the concentration ratio for the calibration curveEq. (1).
ðAVanco=ACeftaÞ ¼ nþm � ½ðVancotheorÞ=ðCeftatheorÞ� ð1Þ
where AVanco is the vancomycin area (mAu); ACefta is the ceftazidime area(mAu); [Vancotheor] is vancomycin theoretical concentration (mg=mL);[Ceftatheor] is ceftazidime theoretical concentration (mg=mL); m is theslope; and n is the ordinate at vancomycin concentration equals 0.
Drug Analysis and Calibration in Pulmonary Tissue
Calibration curves were constructed by cutting small pieces of controllung tissue (from pigs without antibiotic treatment). The observedpeak-area ratio method with reference to the internal standard was appliedto assess drug concentration.
Calculation of the unknown vancomycin concentration in tissue sam-ples was performed using the linear regression equation of the peak arearatio against the concentration ratio for the calibration curve (Eq. (2)).Correction for the tissue experimental weight was taken into account. Astandard equation to normalize weights of pulmonary tissues and theextracted volume after the homogenization process was used.[13]
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f½ðA0Vanco=A
0CeftaÞ=gexp� � VRealg ¼ nþm� f½ðVanco0theorÞ=ðCefta0theorÞ�
� ½gexp=gtheor� � VRealg ð2Þ
where: A0Vanco is the vancomycin area (mAu); A0
Cefta is the ceftazidime area(mAu); [Vanco0theor] is the vancomycin theoretical concentration (mg=mL);[Cefta0theor] is the ceftazidime theoretical concentration (mg=mL); gexp isthe pulmonary pig tissue experimental weight (g); gtheor is the pulmonarypig tissue theoretical weight (0.5 g); VReal is the real volume (L); m is theslope; and n is the ordinate at vancomycin concentrations equals 0.
Sample Collection
Samples were obtained from a model of MRSA pneumonia,[14,15]
developed in mechanically ventilated piglets that were treated with vanco-mycin. All plasma samples were centrifuged at 5000 g for 10min. Plasmawas separated and stored at �40�C until analysis.
In the case of tissue samples, they were freshly cut into pieces ofapproximately 0.5 g and kept frozen at �40�C until analysis. Prior to analy-sis, they were homogenized in a manual homogenator supplied by ANORSA(Barcelona, Spain).
Preparation of Standard and Quality Controls
Buffer Solution (1�TBE Buffer)A 1�TBE buffer solution was prepared by dilution of 10�TBE (1M
Tris, 0.9M Boric Acid, 0.01M EDTA) in ultrapure water (1=10). This sol-ution was maintained at room temperature.
Stock Standard and Quality Control SolutionsThe same stock solution containing 50mg=mL of vancomycin was used
to prepare both standard and quality control material. From this stock sol-ution, three levels of quality control (working standard solutions) at6.25 mg=mL, 25mg=mL, and 100 mg=mL of vancomycin were prepared byappropriate dilutions in free-drug plasma or 1�TBE for tissue samples.These dilutions were spiked with ceftazidime to achieve a final concen-tration of 50mg=mL of internal standard.
Standard PreparationPlasma control specimens were spiked with the appropriate volume of a
stock vancomycin solution to attain several standard samples of differentvancomycin concentrations, and with 175 mL of (IS). Finally, 140mL of
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drug-free plasma was added to obtain a final volume of 350 mL for stan-dards. These standards were mixed and afterwards the mixture was precipi-tated with 350mL of HClO4 3% (1=1, v=v).
Tissue control specimens (0.5 g) were spiked with the appropriate vol-ume of a stock vancomycin solution to attain several standard samples ofdifferent vancomycin concentrations and with 175 mL of ceftazidime stan-dard solution (100mg=mL). One-hundred-forty microliters of 1�TBE wereadded to obtain a final volume of 350 mL for standards, quality control, andunknown samples. These standards were homogenized for 30 sec and thenextracted and precipitated with 350 mL of HClO4 3% (1=1, v=v).
To finish, both matrices were centrifuged at 5000 rpm for 10min and100 mL of the supernatant was directly injected into the chromatographicsystem.
Sample PreparationAll pig plasma samples were spiked with the IS at 50mg=L. This mixture
was precipitated with 350 mL of HClO4 3% (1=1, v=v).All tissue samples (0.5 g) were added with 350 mL of 1�TBE spiked with
the internal standard at 50mg=L. These specimens were homogenized for30 sec, extracted and precipitated with 350 mL of HClO4 3% (1=1, v=v).
Afterward plasma and tissue specimens were centrifuged at 5000 rpmfor 10min and 100 mL of the supernatant was injected into the chromato-graphic system.
Validation Criteria
In accordance with the US Food and Drug Administration’s Guidancefor Industry Bioanalytical Method Validation, and the European Agency forthe evaluation of Medicinal Products’ Guideline on Validation of AnalyticalProcedures, the following seven criteria were evaluated: linearity, accuracy,precision, recovery, limits of detection and quantitation, and stability.[16–19]
In addition, in this study, quality control samples were analyzed in parallelto establish the suitability of the method.
Linearity for Plasma
During method validation, the range of linearity was established basedon three separate runs of assay of freshly prepared calibration standards. Aseven-point standard curve of vancomycin was constructed by drawing theratio between the peak area of vancomycin and the peak area of ceftazi-dime versus the ratio between vancomycin concentration and ceftazidimeconcentration. The concentration of vancomycin ranged from 1.6 to100 mg=mL (Eq. (1)).
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Linearity for Pulmonary Tissue
Calibration curves were constructed by cutting small pieces of controllung tissue (from pigs without antibiotic treatment). A six-point standardcurve of vancomycin was constructed by drawing the ratio between the peakarea of vancomycin and the peak area of ceftazidime versus the ratiobetween vancomycin concentration and ceftazidime concentration. Theconcentration of vancomycin ranged from 3.13 to 100 mg=mL.
Calculation of the unknown vancomycin concentration in tissue sam-ples was performed using the linear regression equation of the peak arearatio against the concentration ratio for the calibration curve. A standardequation to normalize the weights of pulmonary tissues and the extractedvolume after the homogenization process was used (Eq. (2)).
Linearity was assessed in pulmonary plasma and tissue methods,respectively, using Pearson’s coefficient (r), the coefficient of correlation(r2), and the (tstudent) for the slope and Pearson’s coefficient.
Accuracy and Precision
Accuracy of an analytical method is defined as the agreement betweenresults obtained by the analytical method to the true value. Precisiondescribes the degree of the similarity.[16–19] The accuracy of the methodswas performed using a recovery study with fifteen drug spiked samples atthree concentrations (100, 25, and 6.25 mg=mL for plasma and tissuemethod, respectively) measured in one run performed in a single day. Thisallowed us to determine intra-day precision and accuracy.
Analysis of the same drug concentrations over three consecutivedays was performed to determine inter-day precision. Accuracy [ER(%)]and precision [CV(%)] were calculated with equations (Eqs. (3), (4)),respectively):
ERð%Þ ¼ ð½Vanco�exp � ½Vancotheor�Þ=½Vancotheor� � 100 ð3Þ
where ER(%) is Accuracy; [Vancotheor] is Vancomycin theoretical concen-tration (mg=mL); and [Vancoexp] is Vancomycin experimental concen-tration (mg=mL).
CVð%Þ ¼ ðSD=XaveÞ � 100 ð4Þ
where CV (%) is the coefficient of variation; SD is the standard deviation;and Xave: is the average drug concentration (mg=mL).
For the analysis to be considered acceptable in both methods, no morethan one-third of the quality control tests were allowed to deviate from the
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nominal concentration by more than 15%, and at least 50% of the resultsfrom each quality control sample had to be within 15% of the nominalconcentration.
Recovery in Plasma
Recovery was determined by comparison of the analytical results forcalibration curves (mean slope) with calibration curves of unextracted stan-dards (100% recovery). Recovery was calculated with Eq. (5).
Rð%Þ ¼ ðm0VancoPlasma=m
0VancoUnxÞ � 100 ð5Þ
where m0VancoUnx is the mean slope for calibration curves of unextracted
standards (mAu* s); and m0VancoPlasma is the mean slope for calibration
curves of plasma standards (mAu* s).
Recovery in Pulmonary Tissue
Recovery was determined by comparison of the analysis between sam-ples with known concentrations of the analyte and extracts, obtained afterhomogenization of a piece of control pulmonary tissue, spiked with vanco-mycin (plus the IS) at the same concentrations as those prepared in thequality controls. Recovery was calculated by Eq. (6).
Rð%Þ ¼ ðAVancoExp=ACeftaExpÞ=ðAVancoCont=ACeftaContÞ � 100 ð6Þ
where AVancoExp is the vancomycin area of the spiked extract (mAu);ACeftaExp is the ceftazidime area of the spiked extract (mAu); AVancoCont isthe vancomycin area of the control (mAu); and ACeftaCont is the ceftazidimearea of the control (mAu).
Limit of Detection (LOD)
The LOD is determined by the analysis of samples with known concen-trations of analyte and by establishing the minimum level at which the ana-lyte can be reliably detected. Determination of the signal-to-noise ratio isperformed by comparing measured signals from samples with known lowconcentrations of analyte with those of blank samples and establishingthe minimum concentration at which the analyte can be reliably detected.A signal-to-noise ratio 3:1 is generally considered acceptable for estimatingthe detection limit.[16–19]
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Lower Limit of Quantitation (LLOQ)
The LLOQ is the lowest amount of analyte in a sample which can bedetermined with precision and accuracy that is not less than �20%.[16–19]
This parameter was established with the analysis of six or seven sampleswith known concentrations of vancomycin (linearity assay) in plasma o pul-monary tissue, in that order. Bias (ER%) and precision (%CV) at the LLOQwere evaluated in both matrices.
Stability
Freeze and Thaw Cycles for Stability in Plasma and Pulmonary TissueVancomycin stability was determined after three freeze (�40�C) and
thaw cycles of three drug-spiked samples with concentrations of 100 mg=mL, 25mg=mL, and 6.25 mg=mL, all measured in one run performed inone day.
Post-Preparative Stability in Plasma and Pulmonary TissueThe stability of processed samples at room temperature, including the
resident time in the auto-sampler was determined using three aliquots withdifferent concentrations. We compared the results obtained with freshsample with those obtained with these same samples after 24 hr in theauto-sampler.
Stability (S) was calculated with Eq. (7).
Sð%Þ ¼ ðAVanco=j=ACefta=jÞ=ðAVancoCont=ACeftaContÞ � 100 ð7Þ
where AVanco=j is the vancomycin area; j is the three freeze (�40�C) andthaw cycles or 24hr in the auto-sampler extract (mAu); ACefta=j is the cefta-zidime area of the spiked extract (mAu) at the aforementioned scenarios(j); AVancoCont is the vancomycin area of the freshly extracted control(mAu); and ACeftacont is the ceftazidime area of the freshly extractedcontrol (mAu).
The non-parametric Wilcoxon test was used to compare results andp< 0.05 was used for statistical significance. Statistical analysis was per-formed with SPSS 15.0 for windows (SPSS Inc., Chicago, IL, USA).
Pig Plasma Samples
Plasma samples were obtained from a piglet model of MRSA pneu-monia.[14,15] Animals were treated with 15mg=kg (mean dose: 500mg) ofvancomycin twice daily for 96 hr or until death. Blood samples werescheduled to be drawn at several times throughout all the experiment
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(96 hr). Samples were centrifuged at 5000 g for 10min. Plasma was sepa-rated and stored at �40�C until analysis.
Pig plasma samples were spiked with the IS (ceftazidime) at 50mg=L.This mixture was precipitated with 350 mL of of HClO4 3% (1=1, v=v), cen-trifuged at 8000� g for 10min and 100mL of the supernatant was injectedinto the chromatographic system.
Pig Pulmonary Tissue Samples
Lung tissue samples were obtained from the aforementioned pigletmodel of MRSA pneumonia.[14] At euthanasia (at least 12 hr after the lastdose of vancomycin), pulmonary tissue was obtained from several lunglobes and kept frozen at �40�C until analysis.
Tissue specimens (0.5 g aprox.) were spiked with 175mL of ceftazidimestandard solution (100mg=mL). Finally, 175mL of 1�TBE was added toobtain a final volume of 350mL for all samples. These samples were homoge-nized for 30 sec, extracted and precipitated with 350mL of HClO4 3% (1=1,v=v). Afterward, samples were centrifuged at 8000� g for 10min. Finally,100mL of the supernatant was injected into the chromatographic system.
Statistical Analysis
Quantitative variables were expressed as mean� standard error (SE).Data between groups were compared using the Friedman nonparametrictest. A p-value< 0.05 was considered statistically significant. All reportedp values are the exact p values derived through two-tailed tests.
RESULTS
Plasma HPLC Assay
Vancomycin and the internal standard were quantitatively extractedfrom pig plasma after precipitation. Under the experimental conditionsdescribed in the previous section, vancomycin and the IS were elutedwithin the retention windows of 17–18min and 10–11min, respectively.Representative chromatograms of drug-free pig plasma and quality controlsamples are shown in Figure 1a and 1b.
Tissue HPLC Assay
Vancomycin and the internal standard were quantitatively extractedfrom pulmonary tissue after homogenation and precipitation. They were
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eluted within the retention windows of 19–20min and 10–11min, respect-ively. Representative chromatograms of blank tissue and quality controlsamples are shown in Figure 2a and 2b.
Plasma HPLC Assay Validation
The correlation between drug concentration and peak area wasexcellent for human plasma across the concentration range studied (1.6–100 mg=mL). Calibration curves (N¼ 3), the correlation coefficient (r2)and their Pearson coefficient (r) were calculated. They were: y¼ 0.6304x�0.0231 (r2¼ 0.9983; r¼ 0.9991); y¼ 0.6408x� 0.0063 (r2¼ 0.9985;r¼ 0.9992) and y¼ 0.6781x� 0.0160 (r2¼ 0.9965; r¼ 0.9982). In these
FIGURE 1 Chromatogram of (a) drug-free plasma and (b) spiked control vancomycin plasma(50mg=mL).
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equations,� represents the concentration ratio and y the peak area ratio.Pearson coefficient was not significantly different from the unity line(p> 0.05) in any case. Accuracy and precision were assessed by analyzingquality control samples. Results are shown in Table 1.
FIGURE 2 Chromatogram of (a) blank lung tissue and (b) spiked control vancomycin tissue(50mg=mL).
TABLE 1 Results for Validation Parameters for Plasma’s Method
6.25mg=mL 25mg=mL 100mg=mL
Intra-day accuracy (ER%) (n¼ 5) 103.9 94.2 102.1Inter-day accuracy (ER%) (n¼ 15) 102.6 93.4 105.5Intra-day precision (CV%) (n¼ 5) 5.3 3.8 2.6Inter-day precision (CV%) (n¼ 15) 5.9 6.3 3.9
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The LOD was 0.9mg=mL. The LLOQ was 1.6mg=mL (bias andprecision: ER(%)¼ 2.4% and CV(%)¼ 2.6%, respectively). Typical chro-matograms of the LLOQ are shown in Figure 3a and 3b. The meanrecovery remained fairly constant (93.4%). Results from the stabilityanalysis revealed no loss of vancomycin after three freeze (�40�C) andthaw cycles. Results are shown in Table 2. No significant differenceswere seen in vancomycin and ceftazidime chromatographic signals aftermaintaining vancomycin controls for 24 hr in the auto-sampler. S(%)¼95.3% at 100 mg=mL; S(%)¼ 92.5% at 25mg=mL; S(%)¼ 100.1% at6.25 mg=mL.
FIGURE 3 Chromatogram of spiked vancomycin plasma. (a) Lower limit of quantification (LLOQ)including (b) a zoom of the region of interest.
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Tissue HPLC Assay Validation
The correlation between drug concentration and peak area was excel-lent for TBE in tissue across the concentration range studied (from3.1 mg=mL to 100 mg=mL). Pearson coefficient (r) and coefficient ofcorrelation (r2) were calculated: r¼ 0.9978 and r2¼ 0.9958. The regressionline (y¼ 0.0082þ 0.8619x) was not significantly different from the unityline (p> 0.05). Accuracy, precision, and recovery were assessed by analyzingquality control samples. Results are shown in Table 3.
The limit of detection (LOD) was 1.6 mg=mL. The lower limit of quanti-fication (LLOQ) was 3.1mg=mL (bias and precision: ER(%)¼ 9.6% andCV(%)¼ 8.7%, respectively). Typical chromatograms of the LLOQ areshown in Figure 4a and 4b.
The mean recovery, calculated from the ratio of the areas between van-comycin and the IS (AVanco=ACefta), remained fairly constant. It showedvalues of 90.9%, 91.7%, and 91.3% at 100, 25, and 6.25 mg=mL of vanco-mycin, respectively.
Results from the stability analysis revealed no loss of vancomycin afterthree freeze (�40�C) and thaw cycles. Results are shown in Table 4. Nosignificant differences were seen in vancomycin and ceftazidime chromato-graphic signals after maintaining vancomycin controls for 24 hr in theauto-sampler. S(%)¼ 90.6% at 100 mg=mL; S(%)¼ 91.5% at 12.5 mg=mL;S(%)¼ 104.6% at 3.1 mg=mL.
TABLE 2 Results from the Stability Analysis of Vancomycin in Pig Plasma After ThreeFreeze (�40�C) and Thaw Cycles
AVanco=ACefta at Baselinea AVanco=ACefta after 3Cycle
a pb
100 mg=mL 1.33 1.34 0.1125 mg=mL 0.28 0.29 0.116.25mg=mL 0.08 0.08 0.29
aVancomycin area=Ceftazidime area.bp< 0.05 to be significant.
TABLE 3 Results for Validation Parameters for Tissue’s Method
6.25mg=mL 25mg=mL 100mg=mL
Intra-day accuracy (ER%) (n¼ 5) 108.8 92.1 95.5Inter-day accuracy (ER%) (n¼ 15) 106.5 90.8 93.5Intra-day precision (CV%) (n¼ 5) 7.0 5.9 4.1Inter-day precision (CV%) (n¼ 15) 7.8 8.5 5.1
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Pig Plasma and Pulmonary Tissue Samples
The median [range] serum vancomycin concentration at sacrifice(trough) in the group of animals treated with vancomycin every 12 hr were17 mg=mL [7–28 mg=mL; N¼ 8].
FIGURE 4 Chromatogram of spiked vancomycin lung tissue. (a) Lower limit of quantification (LLOQ)including (b) a zoom of the region of interest.
TABLE 4 Results from the Stability Analysis of Vancomycin in Lung Tissue After ThreeFreeze (�40�C) and Thaw Cycles
AVanco=ACefta at Baselinea AVanco=ACefta after 3Cycles
a pb
100mg=mL 0.99 0.97 0.1125mg=mL 0.30 0.29 0.596.25mg=mL 0.08 0.08 0.29
aVancomycin area=Ceftazidime area.bp< 0.05 to be significant.
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Lung samples (from eight piglets) were collected at euthanasia after72–96hr of treatment, and 12 hr after the last drug dose. Median (range)vancomycin trough concentrations in lung tissue at the end of the studyresulted to be 9.6 (8.4–10.7) mg=mL.
DISCUSSION
Specific HPLC methods with adequate specificity, sensitivity, precision,and accuracy have been developed in this study to measure vancomycin inplasma and lung tissue. These methods allow rapid quantitation of vanco-mycin concentrations in plasma and pulmonary tissue, offering a highlysimplistic approach to sample preparation. They are accurate, reproduc-ible, and specific for vancomycin and ceftazidime (SI).
There are several HPLC methods already published in the literature toquantify this drug either in serum or plasma by HPLC-UV.[5,6,9–12,20,21]
Some of these techniques use procedures for sample preparation, includ-ing solid-phase extraction.[5,8] or organic precipitation with acetonitrileor methanol.[9,10] Compared to other procedures, the present method-ology describes a very simple method using precipitation and furtherHPLC-UV to quantify vancomycin in pig plasma. The main advantage ofour method over those previously published lies in the simple preparationof the samples since there is no need for solid phase extraction orliquid-liquid extraction with organic compounds. Our HPLC assay uses asimple single-step sample preparation and common HPLC equipment (fastand reproducible strategy), which could lead to an easy implementation inany bioanalytical laboratory.
The method described here is simple and fast without loss of robust-ness, selectivity, sensitivity, precision, linearity, accuracy, or stability.
Only a few HPLC methods consider vancomycin quantification intissues UV.[5–9]One of them is described by Beckmann et al.[9] They pro-pose an HPLC technique in which 200–250mg of pulmonary tissue ishomogenized and extracted with 8 volumes (w=v) of 1% ortho-phosphoricacid=acetonitrile (30:70, v=v). In this method, the column temperature ismaintained at 30�C in isocratic conditions. Precision and accuracy resultswere better than 8% in a total of 3 lung specimens assayed in duplicated.The recovery is 70% for lung tissue specimens and linearity has beenassayed between (1–10 mg=g). Our study in pulmonary tissue shows betterresults than those studies previously quoted, since, in our case, the intra-dayprecision (of five replicates at three different concentrations) is lower than7%, in all cases, and the inter-day precision (of five replicates analyzed in 3consecutive days) results to be lower than 8.5%. Our recovery is around91% and the linearity has been fixed at 3.1–100 mg=mL. Moreover, data
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from another study, in which lung samples are treated with a 1mL of coldwater, centrifuged twice at 12550 g and the supernatant applied to C18 SPEcartridges (solid-liquid extraction) prior to inject 50mL of the ultrafilteredinto the HPLC system, showed results quite similar to ours. Their cali-bration curve is lineal in the range 0.4–40mg=mL and accuracy is 106%and 109% for 2 and 20mg=L, respectively.
Our extraction method with 1�TBE is simpler, faster, and more econ-omic than the aforementioned assays in pulmonary tissue samples, since:(1) it is not necessary to prepare a mix of two compounds [1%ortho-phosphoric acid=acetonitrile (30:70, v=v)] in the extraction phaseand (2) it uses an easy protein precipitation with HClO4 3%. The assay isalso specific given that no interfering peaks (endogenous or exogenouscompounds) are seen, and data shows a better recovery (91.3� 0.4%) thanthe one reported by Luzzati et al. using SPE cartridges.[8]
Thus, the present method could easily be used in laboratories to deter-mine antibiotic concentrations in pulmonary tissue samples.
A possible drawback of our HPLC method is the fact that our LLOQcould not drop below 3.1mg=mL. However, it should not have any impactin terms of clinical issues since this value is below the minimum inhibitoryconcentration of MRSA for vancomycin for sensitive specimens. Anotherlimitation of the present study could be the further clinical interpretationof the homogenate drug concentration related to the antibiotic levels at thesite of infection. Drug concentrations in tissue may depend on (1) theamount of blood in the tissue sample, (2) drug chemical degradation dur-ing processing, and (3) sample collection. Certainly, blood present in thevessels of the tissue or in its surface is included in the final homogenateand to reduce a possible bias due to this fact, an adjustment by weight ofthe real analyzed portion of lung and the extracted volume after homoge-nation were both taken into account in this analysis. Calibration with inter-nal standard (ceftazidime) was used in this analysis to control any specimenloss (chemical degradation) occurred during the analysis. Furthermore, tocircumvent any artifact or loss of the drug during sample preparation, allstandards were prepared in the appropriate tissue homogenate (lunghomogenate). To avoid the drawback related to sample collection all tissuesamples were taken when the distribution phase of the drug had been com-pleted (3–4hr after dose administration) and the equilibrium between thevascular space and the tissue was fully achieved.
Homogenization implies a disruption of cell membranes and mixesboth intracellular and extracellular fluids and solids, but, in this particularcase, a minor dilutional effect on vancomycin concentration couldbe expected after tissue homogenization, since the drug is mainly distribu-ted into the extracellular space and presents a poor penetration intotissues.
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The availability of an HPLC method to determine antibiotic concentra-tions, both in serum and lung, allows to asses tissue drug penetration whichcould be crucial information to predict clinical response to specific treat-ments. Studies in animal models of pneumonia, where it is possible toobtain lung tissue, offer an exceptional opportunity to learn about thepharmacokinetics (PK), and pharmacodynamics (PD) of antibiotics. Thecombination of PK and PD data might help to further implement optimalantibiotic dosing regimens in humans.
CONCLUSION
The HPLC assays developed in this study allow for rapid, simple,reliable, and inexpensive analysis of vancomycin in plasma and pulmonarypig tissue. Vancomycin analysis, performed using an isocratic mode, is com-pleted within 20min in both methods. These methods might be helpful todevelop future pharmacokinetic studies of vancomycin penetration inpulmonary tissue.
ACKNOWLEDGMENTS
This study was funded by grants from Ciber de Enfermedades Respira-torias (CibeRes, CB06=06=0028) from Instituto de Salud Carlos III (ISCIII),Fondo de Investigaciones Sanitarias (FIS) beca FIS PI070419 and 2009SGR-911.
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