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Journal of Chromatography B, 965 (2014) 100–106

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom epage: www.elsev ier .com/ locate /chromb

atrix effect in F2-isoprostanes quantification by HPLC–MS/MS: Aalidated method for analysis of iPF2�-III and iPF2�-VI in human urine

eresa Petrosino, Mauro Serafini ∗

unctional Food and Metabolic Stress Prevention Laboratory, Agriculture Research Council, CRA-NUT, Rome, Italy

r t i c l e i n f o

rticle history:eceived 8 March 2014ccepted 17 June 2014vailable online 25 June 2014

eywords:iquid chromatographyass spectrometry

2-isoprostanesrineatrix effect

a b s t r a c t

Liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) has become the methodof choice for analysis in biological matrices, because of its high specificity and sensitivity. However, itshould be taken into account that the presence of matrix components coeluting with analytes mightinterfere with the ionization process and affect the accuracy and precision of the assay. For this reason,the presence of a “matrix effect” should always be evaluated during method development, above allin complex matrix such as urine. In the present work, a HPLC–MS/MS method was developed for thequantification of urinary iPF2�-III and iPF2�-VI. A careful assessment of matrix effect and an accuratevalidation were carried out, in order to verify the reliability of quantitative data obtained. Ion suppression,due to the matrix components, was reduced through optimization of both chromatographic method andsample extraction procedure. Urine samples were purified by solid phase extraction (SPE) and the extractsinjected into the HPLC–MS/MS system, equipped with a TurboIonSpray ionization source operated innegative ion mode (ESI−). Stable isotope-labeled analogues (iPF2�-III-d4 and iPF2�-VI-d4) were used as

internal standards, and quantification was performed in multiple reaction monitoring (MRM) mode bymonitoring the following mass transitions: m/z 353.4 → 193.2 for iPF2�-III, m/z 357.2 → 197.0 for iPF2�-III-d4, m/z 353.4 → 115.1 for iPF2�-VI, and m/z 357.4 → 115.1 for iPF2�-VI-d4. The validated assay, appliedto the analysis of urinary samples coming from healthy and overweight subjects, resulted suitable for anaccurate quantification of iPF2�-III and iPF2�-VI in human urine.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Measurement of F2-isoprostanes (F2-IsoP) in plasma and urineas emerged as a reliable approach to assess oxidative stress

n vivo [1,2]. Increased levels of isoprostanes have been reported inatients affected by cardiovascular diseases and related risk factorse.g. smoking, hypercholesterolemia, diabetes, obesity, metabolicyndrome), neurological diseases (e.g. Alzheimer’s disease), lung,enal and liver diseases [3], and cancer [4,5].

F2-IsoP are analogues of prostaglandin F2� (PGF2�), producedn vivo primarily by free radical-induced peroxidation of arachi-onic acid esterified in the sn-2 position of phospholipids [6]. After

heir formation, isoprostanes are released from membrane phos-holipids by the action of phospholipases and excreted in urinehere they are very stable and can be non-invasively measured.

∗ Corresponding author at: CRA-NUT, Via Ardeatina 546, 00178 Rome, Italy.el.: +39 3495742173.

E-mail address: serafini mauro@yahoo.it (M. Serafini).

ttp://dx.doi.org/10.1016/j.jchromb.2014.06.021570-0232/© 2014 Elsevier B.V. All rights reserved.

There are four regioisomeric series of F2-IsoP, named 5-, 8-, 12-and 15-F2-IsoP [7] or also iPF2�-VI, iPF2�-IV, iPF2�-V and iPF2�-III[8], respectively. The basic structures of the four main F2-IsoP arereported in Fig. 1. Among these, the 5- and 15-series are the mostabundant because the intermediates involved in the formation ofthe 8- and 12-series undergo further oxidation. In particular, themost extensively studied F2-IsoP has been 15-F2t-IsoP or iPF2�-III(also known as 8-iso-PGF2�).

Several methods have been developed for isoprostane quan-tification in biological fluids and tissues, and the two mainanalytical approaches adopted have been mass-spectrometry andimmunological techniques. Gas-chromatography coupled to mass-spectrometry (GC/MS) [9] has been considered for a long timethe “golden standard” for isoprostanes measurements. It has theadvantage of high sensitivity and specificity but, at the same time,it requires expensive equipment and time-consuming procedures

for sample preparation, including derivatization of analytes toprotect the polar groups. In order to overcome these drawbacks,alternative methods have been developed using immunologicaltechniques (ELISA, RIA), which have a lower cost and are easy

T. Petrosino, M. Serafini / J. Chromatogr. B 965 (2014) 100–106 101

s of th

tccpussatn

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2

2

df

Su(f

2U

Fig. 1. Basic structure

o perform. However, these commercial immunoassays are sus-eptible to cross-reactivity problems because of which they areonsidered to give only a semi-quantitative estimation of iso-rostanes levels. GC/MS and immunoassays have been extensivelysed for isoprostanes measurements; however, in the last years,everal methods using liquid-chromatography coupled to masspectrometry (HPLC/MS) have been also developed. HPLC/MS is anttractive alternative to GC/MS because it is specific and very sensi-ive and it requires shorter procedures for sample preparation witho derivatization step.

Due to its high specificity, liquid chromatography combinedith tandem mass spectrometry (MS/MS) has become the method

f choice for analysis in biological matrices. It is often thought thatuch selectivity allows simplifying or even eliminating both samplereparation and chromatography. However, it should be consid-red that, although not detected, coeluting compounds mightodify the ion intensity of the analytes and affect the accuracy

nd precision of the assay. By interfering with the ionization pro-ess of analytes, these matrix components can enhance or suppressheir signal; as a consequence, the same amount of analyte mayroduce different signals if measured in either solvent or differ-nt biological matrices. This “matrix effect”, that in most cases isbserved as ion suppression, has been reported to affect the reliabil-ty of quantitative assays using HPLC–MS/MS [10–15]. An efficientxtraction of the analytes and a good chromatography can reduceuch effects, by limiting the coelution of other compounds withhe analytes. However, matrix effect and ion suppression shouldlways be assessed during method development and an accuratessay validation should be carried out. In this work we have devel-ped a validated HPLC–MS/MS method for the quantification ofPF2�-III and iPF2�-VI in humane urine.

. Experimental

.1. Materials

Isoprostane standards (iPF2�-III, iPF2�-III-d4, iPF2�-VI, iPF2�-VI-4) and assay kit for urinary creatinine analysis were purchasedrom Cayman Chemicals (Ann Arbor, MI, USA).

Acetonitrile and methanol, both of HPLC–MS grade, were fromigma–Aldrich (St. Louis, MO, USA). Milli-Q water was preparedsing a Millipore (Bedford, MA, USA) purification system. Ethanol96%), hydrochloric acid (37%) and glacial acetic acid were obtained

rom Carlo Erba (Milano, Italy).

Columns for solid phase extraction (SPE) (Strata C18-E,00 mg/3 ml) were purchased from Phenomenex (Torrance, CA,SA).

e four F2-IsoP series.

2.2. Standards preparation

Calibration standard solutions were prepared in solvent (HPLCmobile phase, A/B = 50/50) at the following concentrations for bothiPF2�-III and iPF2�-VI: 0, 1, 2, 5, 10, 20, 50, 100 ng/ml, correspond-ing to 0, 0.05, 0.10, 0.25, 0.5, 1, 2.5, 5 ng/ml urine equivalent levels.As to internal standards (IS), their concentrations in the calibrationsolutions were 50 ng/ml (corresponding to 2.5 ng/ml urine equiva-lent level) for iPF2�-III-d4 and 10 ng/ml (corresponding to 0.5 ng/mlurine equivalent level) for iPF2�-VI-d4. Calibration curves were con-structed with non-weighted linear regression by plotting the peakarea ratios (analyte area/IS area) against analyte concentrations.

Quality control (QC) samples for method validation were pre-pared at four levels: 1, 2, 10, 50 ng/ml in solvent, and 0.05, 0.10, 0.5,2.5 ng/ml in urine.

2.3. Sample extraction

Urine samples were stored at −80 ◦C. After thawing and vor-texing, different aliquots of each sample (from 0.4 to 2 ml) weretaken according to their creatinine concentration (urinary levels ofcreatinine were previously assessed by using a colorimetric assaykit from Cayman Chemicals). All samples were made up to 2 mlwith water and spiked with 5 ng of iPF2�-III-d4 (final concentra-tion in urine 2.5 ng/ml) and 1 ng of iPF2�-VI-d4 (final concentrationin urine 0.5 ng/ml). Samples were then acidified with 120 �l ofHCl 1 M and subsequently purified through C18 SPE cartridges(Strata C18-E, 200 mg/3 ml, Phenomenex). Columns were condi-tioned with methanol (2.4 ml) and equilibrated with an aqueoussolution of HCl 1 mM (2.4 ml), then the samples were loaded andwashed sequentially with 2.4 ml of HCl 1 mM and 2.4 ml of a HCl1 mM/MeOH = 50/50 solution. Analytes were finally eluted with2.4 ml of a HCl 1 mM/MeOH = 30/70 solution. The eluate was evapo-rated and the residue was dissolved in 100 �l of HPLC mobile phase(A/B = 50/50), filtered and injected into the HPLC–MS/MS system(injection volume 10 �l).

2.4. HPLC–MS/MS instrumentation and conditions

An Applied Biosystems 3200 QTRAP mass spectrometer (Fos-ter City, CA, USA) was used in combination with a Perkin ElmerSeries 200 HPLC system consisting of a thermostated autosam-pler, a binary micro-pump system, a vacuum degasser and acolumn oven. Chromatographic separation was performed on

a Phenomenex Luna C18(2) column (100 mm × 4.6 mm, 3 �m),preceded by a guard column (4 mm × 3.0 mm, 3 �m) and ther-mostated at 25 ◦C. Injection volume was 10 �l and, as mobilephases, were used water with 0.01% of acetic acid (phase A) and

102 T. Petrosino, M. Serafini / J. Chroma

Table 1MS settings.

Source/gasCUR 25.00CAD MediumTEM 600.00GS1 40.00GS2 50.00IS −4500.00

CompoundDP −63.00EP −10.00

aafB4

tmtbi

(33

r

3

3

enciwpd1hwtasatitff

d(ihpama

CE −35 (for iPF2�-III and iPF2�-III-d4)−30 (for iPF2�-VI and iPF2�-VI-d4)

CXP −1.80

cetonitrile/methanol = 80/20 (phase B). Elution was carried out at flow rate of 400 �l/min with the following program: 30% B heldor 1 min, then a linear gradient from 30% B to 70% B in 15 min, 70%

for 2 min, followed by a linear gradient back to 30% B over 3 and min re-equilibration at 30% B.

The flow from the chromatograph was directly introduced intohe TurboIonSpray ionization source, operated in negative ion

ode (ESI −). The source temperature (TEM) was set at 600 ◦C andhe ion spray voltage (IS) at −4500 V; the optimized settings foroth source and compound-dependent parameters were reported

n Table 1.Quantification was performed in multiple reaction monitoring

MRM) mode, by monitoring the following mass transitions: m/z53.4 → 193.2 for iPF2�-III, m/z 357.2 → 197.0 for iPF2�-III-d4, m/z53.4 → 115.1 for iPF2�-VI, and m/z 357.4 → 115.1 for iPF2�-VI-d4.

Instrument control, data acquisition and processing were car-ied out with Analyst software (Applied Biosystems).

. Results and discussion

.1. Method development

Optimization of MS parameters was performed manually forach standard by infusion and flow injection analysis (FIA) tech-iques. In the ESI source operating in negative ion mode, bothlasses of isoprostanes are deprotonated to form [M−1]− precursorons with m/z 353 (or m/z 357 for deuterated analogues). However,

hen tandem mass spectrometry is used to fragment them, specificroduct ions are generated from the two classes. The most abun-ant fragment for iPF2�-III is at m/z 193 while for iPF2�-VI is at m/z15 (Fig. 2). The last one is a specific fragment for class VI isomers;owever, a low percentage of m/z 193 is produced from iPF2�-VI asell (about 10% of m/z 115). For this reason it is important that the

wo classes of analytes do not coelute in order to avoid interferencesnd to obtain reliable results during quantification. As to internaltandards, the most intense fragments were m/z 197 for iPF2�-III-d4nd 115 for iPF2�-VI-d4; in the first case the daughter ion maintainshe chain labeled with deuterium atoms, in the second deuteriums lost during fragmentation. The following mass transitions wereherefore selected to quantify the four analytes: m/z 353.4 → 193.2or iPF2�-III, m/z 357.2 → 197.0 for iPF2�-III-d4, m/z 353.4 → 115.1or iPF2�-VI, and m/z 357.4 → 115.1 for iPF2�-VI-d4.

For HPLC method development, several mobile phases and gra-ients were tested as well as different modifiers for aqueous phaseammonium acetate, formic acid, acetic acid). Ammonium acetates a buffer additive commonly used in negative ionization mode;owever, when we tested this modifier (2 mM added to aqueous

hase), poor chromatograms were obtained. On the contrary, theddition of a low percentage of acid resulted in improved chro-atographic separation and peak shape. Between the two tested

cids, better results were obtained with acetic acid (Ka ∼10−5)

togr. B 965 (2014) 100–106

since the higher strength of formic acid (Ka ∼10−4) decreasedthe peak intensity by inhibiting the ionization in the ESI source.Several percentages of both formic and acetic acid were tested,and a good compromise between separation/peak shape and peakintensity was reached with 0.01% of acetic acid. Regarding organicphase composition, acetonitrile, methanol and their mixtures weretested. Good results were obtained with 100% acetonitrile as faras standard solutions were analyzed; however, when real sampleswere injected, the presence of unknown compound coeluting withisoprostanes was observed. In order to avoid ion suppression dueto these compounds, organic phase composition was changed, byadding different percentages of methanol. This resulted in a higherretention of isoprostanes and in a better separation from interfer-ing substances; the best results in terms of both separation andretention times were obtained with acetonitrile/methanol = 80/20and the above described gradient (see Section 2).

As to urine purification, two different approaches were tested.A liquid–liquid extraction was initially tried, by using ethyl acetateand diethyl-ether as extraction solvents. However, this kind ofapproach did not produce good sample purification. With bothsolvents, a high degree of ion suppression was observed, due tothe presence of matrix interferences. Better results were obtained,instead, with solid-phase extraction (SPE). Two different kinds ofcartridges were tested (C18 and polymeric) during method devel-opment, and the C18 columns were finally selected. Both urinesamples and aqueous phases used during SPE were acidified toensure that isoprostanes were not in dissociated form and werenot lost with aqueous phase washes. In order to eliminate mostof the matrix interferences and obtain a good purification, a washwith 50% of organic phase was required. Elution was then carriedwith 70% of organic solvent.

Optimization of both chromatographic method and sampleextraction greatly reduced ion suppression due to matrix com-ponents. MRM chromatograms of a standard solution containingiPF2�-III, iPF2�-VI and their internal standards (iPF2�-III-d4 andiPF2�-VI-d4) are reported in Fig. 3. As can be seen the two ana-lytes are well separated, with elution time of 12.68 for iPF2�-III and13.52 for iPF2�-VI. The two internal standards elute at the sametime of the corresponding analyte. As previously stated, the frag-ment at m/z 193 is produced in a low percentage from iPF2�-VI too;thus, for the transition m/z 353.4 → 193.2, in addition to the mainpeak at 12.68 due to iPF2�-III, we also observe a smaller peak at13.52 due to iPF2�-VI. The same is observed for m/z 357.2 → 197.0transition in case of deuterated analogue. Fig. 4 shows theMRM chromatograms obtained from a representative urinesample.

3.2. Matrix effect assessment

The presence of “matrix effect” can be evaluated by meansof several experimental procedures. One of these involvespost-column continuous infusion of a standard into the mass spec-trometer while an extract from biological matrix is injected intoHPLC system. Since the analyte is introduced into the spectrom-eter at a constant flow rate, a constant signal should be observed.Actually, when an extract is injected into the HPLC, a suppression ofsignal is commonly observed at different elution times dependingon the kind of compounds present in the matrix. If our analytes elutein a region where ion suppression is not observed, it can be statedthat no matrix effect is present. On the contrary, if this suppressionoccurs at the retention times of the analytes, its effect on the reli-

ability of quantification should be evaluated. The above describedapproach does not allow to quantify the entity of matrix effect; forthis aim another procedure should be used involving comparison ofthe signals obtained with (a) standards injected directly in mobile

T. Petrosino, M. Serafini / J. Chromatogr. B 965 (2014) 100–106 103

ctra of

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Fig. 2. Product ion spe

hase, (b) the same amount of analyte added to pre-extracted sam-les, and (c) the same amount of analyte added to matrix beforextraction [16]. The difference between data (b) and (a) provides

measure of matrix factor (MF) while the difference between (c)nd (b) allows to calculate extraction recoveries (RE). The differenceetween (c) and (a) is therefore an index of the overall process effi-iency (PE). The MF, RE and PE values can be calculated as follows:

F (%) = BA

× 100 (1)

C

E (%) =

B× 100 (2)

E (%) = CA

× 100 = MF × RE100

(3)

iPF2�-III and iPF2�-VI.

where A, B and C are the peak areas for standards in mobile phasesolution, standards spiked after sample extraction and standardsspiked before extraction, respectively.

This approach was used to evaluate the extent of matrix effectin our method. In order to account for endogenous isoprostanes,the peak areas of analytes in unspiked samples were subtractedfrom areas in enriched samples. The matrix factor was calculatedfor each analyte and IS. The ratio between MF of the analyteand MF of its IS, named IS-normalized MF, was also calculated.This ratio should ideally be 1; in fact, if the same matrix effectis observed for analyte and IS, the peak area ratio (analyte/IS)should not change and therefore the quantification should not

be affected by matrix. For iPF2�-III a MF around 75% was foundwhile for iPF2�-VI a higher ion suppression was observed with aMF around 50%. However, since similar values were obtained forthe related internal standards, the IS-normalized MF resulted to be

104 T. Petrosino, M. Serafini / J. Chroma

Fig. 3. MRM chromatograms of a standard solution containing iPF2�-III, iPF2�-VIand their internal standards (iPF2�-III-d4 and iPF2�-VI-d4).

togr. B 965 (2014) 100–106

approximately 1 for both analytes (0.98 for iPF2�-III and 0.96 foriPF2�-VI).

The MF calculated in this manner may be referred to as an “abso-lute” matrix effect since the signal response of the standard presentin the extract is compared to the response of the standard in asolvent. Besides the “absolute” matrix effect, it is important to eval-uate the presence of a “relative” matrix effect, by comparing theMF values obtained from different lots of bio-fluids. The presenceof a relative matrix effect can be also evaluated by comparing theslopes of standard lines constructed in different lots of matrix [16].Since slopes are calculated from linear regression analysis of peakarea ratios of analyte/IS versus analyte concentration, their vari-ability is indicative of the real effect of matrix on quantification.According to Matuskewzki et al. [16] if the slopes calculated in fivedifferent sources of bio-fluids are practically the same (CV < 4–5%),any significant matrix effect on quantification may be consideredabsent. If the standard lines are constructed by spiking the sam-ple after extraction, the variability of their slopes depends only onthe matrix effect; if the standard lines are, instead, constructed byspiking the sample before extraction, then variability of their slopeswill reflect the combined effect of matrix and extraction recoveries.Following this procedure we prepared three sets of five standardlines: the first (set 1) in the mobile phase, the second (set 2) inurine extracts originating from five different sources and spikedafter extraction, and the third (set 3) in the same urine samples ofset 2 but spiked before extraction. The variability of the slopes ofeach set was assessed and the CV resulted to be <5%, confirmingthe absence of relative matrix effect.

3.3. Method validation

The assay was validated according to the recent guideline on bio-analytical method validation of European Medicines Agency (EMA)[17].

3.3.1. Sensitivity and linearityThe lower limit of quantification (LLOQ) was determined as the

lowest concentration of the calibration curve for which accuracyand precision ≤20% was obtained [17,18]. According to guidelines[17,18], calibration curves should be prepared in the same biologi-cal matrix as the samples in the intended study by spiking the blankmatrix with known concentration of the analyte. However, blankmatrices were not available for isoprostanes quantification sincethey are endogenous compounds. Therefore, during method devel-opment we compared the curves prepared in urine samples withthe ones prepared in solvent. Because the slopes obtained for thetwo sets of standard lines were not statistically different (pairedt-test), preparation of curves in mobile phase was the standardprocedure adopted for the validated method. The assay was lin-ear over the working range from 1 to 100 ng/ml, corresponding to arange of 0.05–5 ng/ml in urine (R2 > 0.999). LLOQ was set at 1 ng/ml(0.05 ng/ml urine equivalent levels) for both iPF2�-III and iPF2�-VI.

3.3.2. Accuracy and precisionAccuracy and precision were assessed by the analysis of QC sam-

ples prepared at four levels: 1 (LLOQ), 2, 10, 50 ng/ml in solvent,and 0.05 (LLOQ), 0.10, 0.5, 2.5 ng/ml in urine. Five samples per levelwere used, and both intra-day and inter-day accuracy and preci-sion were evaluated according to EMA [17]. For accuracy the meanvalue obtained with QC should be within 15% of the nominal value,except for the LLOQ, which should be within 20% of the nominalvalue. In the same manner, for precision the CV% at each concen-

tration level should not exceed 15%, except for the LLOQ where itshould not exceed 20%. The results obtained during accuracy andprecision assessment met all the established criteria [17]. The accu-racy and precision data in urine samples are reported in Table 2. For

T. Petrosino, M. Serafini / J. Chromatogr. B 965 (2014) 100–106 105

Fig. 4. MRM chromatograms of a urine extract.

Table 2Intra-day and inter-day accuracy and precision in urine samples.

Standard Spiked amount (ng/ml) Intra-daya Inter-dayb

Accuracy (%) Precision (CV%) Accuracy (%) Precision (CV%)

iPF2�-III

0.05 84.5 8.8 86.1 10.90.1 89.8 1.8 87.0 3.40.5 112.6 2.8 109.8 1.62.5 103.1 4.3 96.4 5.5

iPF2�-VI

0.05 87.0 7.5 85.7 8.20.1 92.5 1.6 94.3 1.40.5 105.0 2.6 108.2 5.82.5 90.7 3.2 102.9 2.7

a n = 5/concentration.b Samples analyzed on three different days; n = 5/concentration and day.

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06 T. Petrosino, M. Serafini / J. C

ccuracy assessment, the concentration of QC samples prepared inrine was corrected taking into account the endogenous analyteoncentration.

.3.3. Matrix effectBoth absolute and relative matrix effect were evaluated, as

escribed in Section 3.2.

.3.4. Extraction recoveriesExtraction recoveries (RE) were calculated for both analytes and

S as previously described: RE (%) = C/B × 100, where B and C arehe peak areas for standards spiked after sample extraction andtandards spiked before extraction, respectively. Peak areas wereorrected by subtracting the endogenous levels of isoprostanesresent in unspiked samples. The mean recoveries obtained were5% for iPF2�-III (92% for iPF2�-III-d4) and 63% for iPF2�-VI (65% for

PF2�-VI-d4).

.3.5. Stability studiesStability studies were carried out according to EMA [17] at two

ifferent levels (low and high). Stability of analytes was evaluatedn working solutions using QC samples at 2 and 50 ng/ml, and inrine samples at the corresponding levels of 0.1 and 2.5 ng/ml.oth short-term stability (24 h) at room temperature and long-erm stability (3 and 6 months) at storage conditions (−80 ◦C) weressessed, as well as stability after three freeze–thaw cycles. Thenalytes were stable under the applied conditions, with concentra-ions remaining within 15% of the nominal value.

.3.6. Carryover effectCarryover was assessed by analyzing urine sample enriched

ith iPF2�-III and iPF2�-VI at the upper limit of quantification fol-owed by blank methanol samples. No carryover was observed foroth analytes.

.4. Application of the assay

The validated HPLC–MS/MS method was used to quantify iPF2�-II and iPF2�-VI in 20 urine samples, 10 coming from healthyubjects and 10 from overweight volunteers. Ethical consent wasbtained from all subjects for taking urine samples. The con-entration of isoprostanes was normalized to urinary creatinine.n samples obtained from healthy individuals, iPF2�-III levelsanged from 180 to 458 pg/mg creatinine (with an average level of46 ± 98 pg/mg creatinine) and iPF2�-VI levels ranged from 520 to162 pg/mg creatinine (with an average level of 949 ± 190 pg/mgreatinine). Similar results were obtained from urine of over-eight subjects, with iPF2�-III concentration ranging from 202 to

97 pg/mg creatinine (average level of 356 ± 135 pg/mg creatinine)nd iPF2�-VI levels ranging from 569 to 1498 pg/mg creatinineaverage level of 1022 ± 285 pg/mg creatinine).

. Conclusions

In the present work a HPLC–MS/MS method for urinarysoprostane quantification was developed and validated. Bothurification of samples and chromatographic separation were opti-ized in order to reduce the ion suppression due to the matrix.

[

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togr. B 965 (2014) 100–106

Moreover the use of stable isotope-labeled analogues as inter-nal standards contributed to eliminate the matrix effect, since theobserved ion suppression was identical for the analytes and theirdeuterated analogues. It should be considered, however, that theuse of a stable isotope-labeled standard doesn’t solve accuracy andprecision issues in case of elevated ion suppression. If this sup-pression significantly reduces the signal of the analyte or IS, thesignal-to-noise ratio may be compromised with negative effectson accuracy and precision. For this reason ion suppression shouldbe assessed even if an internal standard is used. In our method ionsuppression reported for iPF2�-III was about 25% (MF ∼ 75%), whilefor iPF2�-VI it was around 50% (MF ∼ 50%). However, it did not affectthe accuracy and precision of the assay since the signals for bothanalytes remained above the LLOQ.

In conclusion, the matrix effect should always be evaluated dur-ing validation of quantitative HPLC–MS/MS methods, above all incomplex matrix such as urine. In this work a careful assessmentof matrix effect and an accurate validation were carried out, inorder to verify the reliability of quantitative data obtained. The val-idated HPLC–MS/MS method was therefore used to quantify thetwo main urinary isoprostane classes in a test sample of 20 sub-jects. The assay resulted suitable for an accurate determination ofendogenous iPF2�-III and iPF2�-VI in human urine.

Acknowledgment

To the Project: QUALIFU – Qualità Alimentare e FunzionaleItalian Ministry of Agriculture (MIPAAF) D.M. 2087/7303/09 of28/01/2009 for financial support.

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