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Sensitive rapid fluorescence polarization immunoassay for free mycophenolic acid determination in human serum and plasma Bettina Glahn-Martínez, Elena Benito-Peña, Francesca Salis, Ana B. Descalzo, Guillermo Orellana, and Maria C. Moreno-Bondi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00780 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Analytical Chemistry

Sensitive rapid fluorescence polarization immunoassay for free mycophenolic acid determination in human serum and plasma Bettina Glahn-Martínez†, Elena Benito-Peña*,†, Francesca Salis‡, Ana B. Descalzo‡, Guillermo Orellana‡, María C. Moreno-Bondi*,† † Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid, Spain. ‡ Department of Organic Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Av. Complutense s/n, 28040 Madrid, Spain. ABSTRACT: In this article, we describe a fluorescence polarization immunoassay (FPIA) using a new label - near-infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA, MPA-AO, for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λexcmax) shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 µg/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7–39 ng/mL (20–80% binding inhibition) in PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus and the results compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).

Therapeutic drug monitoring (TDM) of pharmaceuticals with a narrow therapeutic range or window is an issue of increasing interest for the Public Health authorities, since it is an essential tool to optimize individual dosage regimens for optimal patient benefit with minimal adverse effects. Particularly, quantification of immunosuppressant drugs in solid organtransplanted patients is essential since these drugs usually exhibit large inter- and intra-individual absorption variations, causing unpredictable levels in blood.1 In this scenario, TDM analyses help the clinician to individualize drug dosages avoiding an overdose of immunosuppressive drug and consequently a toxic effect, or underdoses resulting in a high rate of relapse. Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) administered to prevent graft rejection. MPA is a secondary metabolite, derived from Penicillium fungus,2,3 that blocks proliferation of lymphocytes (T- and B-cells) and shows anti-bacterial,4 anti-viral, anti-fungal and anti-tumor activity.5 MPA binds efficiently to albumin (97–99%).6 However, most studies demonstrate that only the free drug fraction is pharmacologically active as it can cross the cellular membrane and bind to receptors promoting the immunosuppressive effect.7 As discussed in the literature,8,9 measurement of the free MPA concentration in plasma, serum or whole blood, rather than the total amount, provides a better correlation with the clinical

efficacy of the drug. However, quantification of free MPA in blood is technically challenging.10 Currently, liquid chromatography (LC) with diode array (DAD), fluorescence (FLD), or mass spectrometry (MS) detection are the analytical techniques of choice in clinical laboratories for the determination of free MPA.4,11,12 While all these techniques provide sensitive and reproducible methods, they require properly trained staff, their cost is high and, besides the analysis time for the chromatographic separation, some methods require additional extraction and derivatization steps, further increasing the analysis times. Therefore, they are inadequate for semi-continuous monitoring with point-of-caretesting devices and for high-throughput screening. The development of screening methods based on antibodies has received much attention from both clinical laboratories and diagnostics companies. The main disadvantage of immunoassays (IAs) compared to chromatographic methods is their potential cross-reactivity towards active or inactive metabolites in the sample, which often results in an overestimation of the concentration of the target analyte. However, one of the advantages of IAs is the lack of sample pretreatment, a necessary step in chromatography.13,14 Unlike chromatographic techniques, immunoassays can be better integrated into a core specialized laboratory (automation, traceability and ease of method validation) or even be part of a TDM point-of-caretesting (POCT) device.15 Several immunoassays have been commercialized for the determination of MPA using different

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detection schemes11 such as, the enzyme-multiplied immunoassay technique (Roche EMIT® and EMIT II®)16 and the cloned enzyme donor immunoassay (CEDIA®, Thermo Fisher Scientific),17 along with immunoturbidimetric assays such as particle enhanced turbimetric inhibition immunoassay (PETINIA).18 However, none of these approaches measures the free MPA level in blood. Only a tentative approach to monitor free MPA has been published using a modified Roche EMIT® assay, although the authors reported non-validated results affected by the presence of an “unidentified” matrix interference.19 Other disadvantages of these immunoassays include a relatively poor selectivity −at least for MPA analysis− with several potential interferences.15,16 For example, cross-reactivity with glucuronide metabolites of MPA may yield positive overestimations ranging from 8.3 to 52.3 %.20 Fluorescence polarization (or fluorescence anisotropy) immunoassays (FPIA) offer a powerful alternative for quantification of free MPA in blood samples. FPIA is a type of the homogeneous fluorescence-based immunoassays that has been applied to the determination of immunosuppressant drugs, such as everolimus (Seradyn Innofluor Certican® FPIA)21 or cyclosporin A22 but, to the best of our knowledge, never for MPA analysis. In most cases, a competitive immunoassay is performed where either the antibody-bound or antibody-free fractions of a fluorescently-labeled antigen (Ag*) are indirectly proportional to the amount of target drug in the sample. The advantages of anisotropy-based immunoassays include the measurement of the bound/free ratio without the need of separation and their excellent capability for automation, as demonstrated by the incorporation of the FPIA technology in some commercial analyzers, such as the Abbott IMx® batch analyzer, for the analysis of small molecules of clinical interest.23 One disadvantage associated with conventional FPIA is the relative high detection limits (LODs) obtained for some targets.24 In some cases, this fact can be ascribed to the use of fluorescein as label. Although this fluorophore exhibits a very high emission quantum yield (Φf = 0.93),25 the sensitivity of the assay is low due to its poor photostability, the interference of sample background (i.e. blood plasma or serum) and the light scattering effect, that can be important in the spectral region of fluorescein excitation and emission (493 and 513 nm, respectively, in aqueous basic medium). Replacement of fluorescein with more photostable labels absorbing and emitting in the red-near infrared region (650 – 900 nm) should improve the FPIA detection limits.26,27 Potential candidates are long-wavelength emitting fluorophores such as cyanine dyes. These labels are positively charged molecules with two aromatic or heterocyclic rings linked by a polymethinic chain via two terminal N atoms. However, one of the main drawbacks of these dyes is their poor photostability.28 As alternative to cyanines, the oxazine and benzoxazine dyes –also positively charged fluorophores with two N atoms connected through a conjugated π system– present a more rigid structure, that provides higher photostability and significant Φf at wavelengths above 650 nm. For example, a popular fluorescent benzophenoxazine is Nile Blue (NB), with a Φf of 0.27 in ethanol.29 Gómez-Hens and coworkers have proved the suitability of a NB tag for the detection of the antibiotic amikacin in human serum samples using a fluorescence anisotropy immunoassay30 with excellent sensitivity and minimal interference from the light scattering.

In this work we describe the development of a novel FPIA for determination of free mycophenolic acid in blood samples. The MPA immunosuppressant has been labeled with a ωaminoderivative of a benzo[a]phenoxazine dye (abbreviated AO, Figure S1, Supporting Information), with a chemical structure resemblant of NB. The novel NIR-fluorescent MPAAO conjugate has been chosen according to the following features, essential to the sought application: ease of synthesis and conjugation to the carboxylic group of MPA (via the ωamino alkyl linker of AO); availability and low price of precursors; high photostability; good water solubility; high fluorescence quantum yield and ease of purification. The conjugate has been applied to the development of a competitive FPIA for quantifying free MPA in serum and blood samples. Several parameters affecting the FPIA performance have been optimized, including the concentrations of MPA-AO and the MPA-specific antibody, as well as the assay buffer and incubation times. Finally, the assay has been used to analyze free MPA in the blood of a heart-transplanted volunteer, treated with the immunosuppressing drugs mycophenolate mofetil and tacrolimus, as well as in the blood of a healthy individual spiked with MPA. The results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD). The novel assay shows promise as a basis for further development of free MPA analyzers in POCT systems based on FPIA technology.

MATERIAL AND METHODS Reagents, solvents and consumables. 1-Naphthylamine (99%) and 4-(dimethylamino)pyridine (DMAP, 99%) were from Sigma-Aldrich (St. Louis, MO), 3-bromopropylamine hydrobromide (98%) was from Acros Organics (Geel, Belgium), 3-ethylamino-p-cresol was from by TCI (Zwijndrecht, Belgium). Absolute ethanol (EtOH, HPLC grade) and hydrochloric acid were from Scharlau (Badalona, Spain). N,N’diethylcarbodiimide hydrochloride (EDC) and trifluoroacetic acid (TFA, peptide synthesis grade) were obtained from Fluorochem (Hadfield, Derbyshire, UK). Mycophenolic acid (MPA) was from Alfa Aesar (Karlsruhe, Germany). Mycophenolic acid β-D-glucuronide (MPAG) was supplied by Carbosynth (Compton, UK). Tacrolimus (FK506) and cyclosporin A (CsA) were from Sinoway Industrial (Xiamen, China). Anti-Mycophenolic acid antibody was obtained from Randox Laboratories (Antrim, UK). Human Serum type AB, phosphate buffered saline with Tween 20 (PBST, pH 7.4), 2(N-morpholino)ethanesulfonic acid (MES), Pierce® ProteinFree Blocking Buffer (PFBB), dichloromethane and acetonitrile (ACN, HPLC grade) were from Thermo Fisher Scientific (Rockford, IL, USA). The fluorescence standard Oxazine170 was obtained from Radiant Dyes (Wermelskirchen, Germany). Dimethyl sulfoxide (DMSO, anhydrous) was provided by VWR (Radnor, PA). Amicon® Ultra with Ultracell 3K ultrafiltration membrane was from Merck Millipore (Cork, Ireland). Black 96-well OptiPlates were from PerkinElmer (Waltham, MA). Ultrapure water obtained from a Millipore Milli-Q water purification system was used in all the experiments. MPA and MPA-AO stock solutions were prepared in DMSO at 2 mg/mL (6.24 mM) and 1 mg/mL (1.43 mM), respectively and stored at 4 ºC. MPA standard solutions were freshly prepared daily upon dilution of stock solutions in PBST (10 mM, pH 7.4).


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Analytical Chemistry

Instrumentation. 1H-NMR spectra were recorded on a Bruker Avance DPX 300MHz-BACS60 spectrometer. NMR chemical shifts are expressed relative to the signals of the non-

deuterated traces of the solvent (CD3OD at 3.34 ppm31). Mass spectra were obtained with a Bruker HCT Ultra (ESI). UV-Vis

Figure 1. Workflow of the assay protocol. MPA quantification was based on a competitive assay in which MPA and the MPA-AO conjugate compete for the binding sites of an anti-MPA antibody. As a result of competition, an increase of the rotational freedom of the fluorescent tracer due to a higher concentration of the analyte results in a decrease in polarization signal.

absorption spectra were measured with a Varian Cary 3-Bio spectrophotometer. Steady-state fluorescence measurements were carried out on a Horiba Fluoromax-4TCSPC spectrofluorometer equipped with a 150-W xenon lamp. Fluorescence quantum yields of AO and MPA-AO were determined in ethanol and phosphate buffer pH 7.4 using Oxazine 170 as standard (Φf = 0.579 ± 0.032 in ethanol),32 with excitation at 595 nm. All measurements were carried out in triplicate and absorption was always kept below 0.1 at the absorption maximum. For the FP immunoassays, a BMG LABTECH CLARIOstar (Ortenberg, Germany) microplate reader was used in the fluorescence polarization mode with a 590-50 nm excitation filter, a 639 nm dichroic filter and a 675 nm emission filter. The degree of polarization is expressed in terms of fluorescence polarization (P) and is determined by eq. 1:22 ∥ m 10 10 (1) ∥ where ∥ is the fluorescence intensity parallel to the excitation plane and is fluorescence intensity perpendicular to the excitation plane. Gain and focus adjustments were set on a reference well containing MPA-AO (polarization adjusted to a reference value of 40 mP). Chromatographic analyses were carried out with an Agilent 1200 series Rapid Resolution Liquid Chromatography (RRLC) system (Palo Alto, CA) equipped with a binary pump, online degasser, high performance autosampler, column thermostat and a 1260 Infinity DAD detector system. Chromatographic separation was carried out using an ACE Excel 2 C18-PFP analytical column (100 mm × 2.1 mm, 2 µm) from Advanced Chromatography Technologies (Aberdeen, Scotland). Centrifugal procedures were carried out on a miniSpin microcentrifuge from Eppendorf AG (Hamburg, Germany).

Synthesis of the NIR fluorescent label AO. For synthesizing of the ω-amino oxazine derivative (AO), nitration of commercially available 3-(ethylamino)-p-cresol and aminoal-

kylation of 1-naphthylamine with 3-bromopropylamine were carried out in the first place. Condensation of the two resulting products in refluxing acidic ethanol yielded AO in 64% yield. The conjugation reaction of AO to MPA was performed with EDC activation of the carboxy group in the presence of DMAP (Figure S1, Supporting Information). The MPA-AO product was obtained in 80% yield after separation with water and dichloromethane. More details on the synthesis of AO and MPA-AO are provided in the Supporting Information.

Sample preparation. PBST pre-rinsed 3K Amicon® Ultra ultrafiltration devices (Figure S2 in the Supporting Information) were used to isolate the MPA free fraction from biological samples. To this aim, the biological sample solution was centrifuged at 2000×g for 15 min at room temperature. Then, a 500 µL aliquot of the sample was transferred to the filter device and centrifuged at 12045×g for 30 min; the resulting ultrafiltrate was made up to the initial volume of 500 µL. After a 1/4 dilution with PBST, the solutions were analyzed by FPIA.

Fluorescence polarization immunoassay. The competitive FP immunoassay was carried out with MPA standard solutions or spiked samples, in the presence of MPA-AO and the anti-MPA antibody at the optimized concentrations. Briefly, the MPA-AO conjugate (20 µL, 10 nM) was mixed with 80 µL of either MPA standard solutions or serum samples in a 96well plate, previously blocked with 300 µL/well of PFBB (Figure S3, Supporting information) for 1 h. Then, 100 µL of anti-MPA antibody (10 µg/mL) was added to the solution and, after an incubation period of 20 min at 29 ºC, the FP value was measured. A blank control was also measured using PBST as the sample. The workflow of the assay protocol is shown in Figure 1. The fluorescence polarization data were plotted as the B/B0 ratio (being B and B0 the fluorescence polarization signal in the presence and in the absence of MPA, respectively) against


Analytical Chemistry the MPA concentration on a logarithmic scale as described in the Supporting Information. Cross-reactivity (CR) for other immunosuppressants and mycophenolic acid β-D-glucuronide (MPAG), was determined using the optimized FPIA. The cross-reactivity (CR) values were calculated according to eq. 2.

CR(%)

ICMPA ICcross-react.

× 100

(2)

Analysis of serum samples. Serum samples (1.5 mL) were spiked with MPA at 1, 5 and 10 µg/mL. The samples were vortexed for 3 min and then centrifuged at 2000×g for 15 min. The supernatant (500 µL) was transferred to the Amicon® system and assayed as described above. For method comparison purposes, the ultrafiltered samples were acidified with TFA to a final concentration of 0.1% prior to chromatographic analysis. An isocratic program was used for MPA separation using a mobile phase containing a mixture of water (with 0.1% TFA) and acetonitrile (with 0.1% TFA) (80:20, v/v). Analyses were performed at a flow rate of 0.6 mL/min and a column temperature of 45 ºC. Under these conditions, with an injection volume of 100 µL, MPA was eluted within 9 min. The DAD detector wavelength was set at 304 nm. Quantification was performed using external calibration and peak area measurements.

those obtained for the free label: λabsmax/λemmax of 630/642 nm and 624/677 nm in ethanol and PBS pH 7.4, respectively, with Φf of 0.55 in ethanol and 0.18 in PBS. The shape of the normalized absorption and emission spectra of MPA-AO in both solvents (Figure 2B) displays a good match indicating that the fluorescence label is not prone to aggregation in water at micromolar concentrations. Good solubility is an important feature for performing reproducible bioassays in aqueous media, since NIR dyes are normally large, planar, hydrophobic molecules prone to aggregation in water. Burgess and coworkers have reported the synthesis of water-soluble NB derivatives (at a 4 µM concentration level) bearing water-solubilizing substituents in their structure. 34 These dyes showed similar variations in fluorescence to AO in going from alcohol (MeOH) to PBS solution: λabs/λem of 628/662 nm in MeOH, and 630/671 nm in PBS, and a reduction of Φf from 0.56 in MeOH to 0.14 in PBS.

A

Analysis of blood samples from a transplanted patient. The concentration of free MPA was determined in the

RESULTS AND DISCUSSION Spectroscopic characterization of MPA-AO. Absorption and emission spectra of the labelled immunosuppressant (MPA-AO) are shown on Figure 2B, in both organic media (ethanol) and 100% aqueous phosphate buffer solution at physiological pH. The absorption maxima are centered at 629 nm in both solvents, while fluorescence peaks at 644 and 654 nm in ethanol and PBS, respectively. The emission quantum yield is remarkable in ethanol solution (Φf = 0.48 ± 0.05) and somewhat lower in aqueous PBS (0.16 ± 0.02), yet the fluorescence is still strong enough to allow a sensitive detection. The latter is an important observation, since in spite of the related Nile Blue (NB) dye being a strongly emissive in organic media (Φf = 0.27 in ethanol), its fluorescence quantum yield in aqueous solution drops to only 0.01, due to due to efficient proton transfer from the solvent.33 Actually, the AO tag is closer to Oxazine 720 than to NB in its photophysical properties. The results obtained for MPA-AO are in agreement to

B 0.9

0.9

0.6

0.6

0.3

0.3

0.0

400

500

600 λ (nm)

700

800

F (norm.)

blood of a volunteer 39-year-old heart-transplanted female patient who was receiving an oral dose of mycophenolate mofetil (250 mg twice a day). Blood samples (20 mL) were collected in EDTA-treated tubes after 12 h of the drug administration. Negative control blood from one healthy individual was also collected. Blood samples were centrifuged at 2000×g for 15 min at room temperature for the plasma isolation. Then, 500 µL of the supernatant was added to PBST pre-rinsed ultrafiltration 3K Amicon® Ultra tubes and centrifuged at 12045×g for 30 min. The plasma extracted samples were stored at -20 ºC until further analysis. Measurements of free MPA in plasma were performed by FPIA, after a 1/4 dilution with PBST, and the results were compared with those obtained by RRLC-DAD after acidification with TFA (final content 0.1%) and no further dilutions.

A (norm.)

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0.0

Figure 2. [A] Chemical structure of the MPA-AO and MPA. [B] Normalized absorption and fluorescence spectra of MPA-AO (4 µM) in ethanol (solid black line) and in PBS at pH 7.4 (dashed red line).

The suitability of MPA-AO for FP assays was evaluated by performing a saturation binding analysis with a fixed concentration of the fluorescent conjugate (1 nM) and increasing concentrations of human serum albumin (HSA) as a model protein to which MPA reportedly binds to a significant extent.4 As shown in Figure S4 (Supporting Information), the FP of MPA-AO increases 10-fold up to ca. 250 mP when fully bound to the protein. This value (the theoretical maximum for a randomly oriented bound fluorophore is 400 mP)23 shows that the fluorescent AO tag does not hinder binding of MPA, while its tumbling is restricted enough to yield a high polarization when the conjugate is bound.

Optimization of the tracer and Ab dilutions. One of the limiting factors for the use of fluorescently-tagged antigens


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Analytical Chemistry in immunoassays is often the poor recognition of the labeled guest by the paratope of the corresponding antigen-specific antibody.22 Thus, the avidity of MPA-AO for the anti-MPA antibody was evaluated, and its specificity was tested by determining its binding to antibodies selective to other immunosuppressants, such as anti-tacrolimus and anti-cyclosporin A. As it is shown in Figure 3, the FP measured with the MPAspecific antibody (25 µg/mL) for the labeled antigen (1 nM) in PBST buffer is actually higher than that obtained in the binding of the former to a model protein (see previous section), and no cross-reactivity was observed with the antibodies specific to tacrolimus or cyclosporin A.

Figure 4. Effect of the buffer composition on the B250/B0 ratio (open symbols). PBST: phosphate buffer (10 mM, pH 7.4) with Tween 20 (0.05%); PFBB: Pierce® Protein Free (PBS) Blocking buffer; BCT: carbonate buffer (10 mM, pH 9.0) with Tween 20 (0.05%); MEST: MES buffer (10 mM, pH 5.7) with Tween 20 (0.05%). The blue bars correspond to the fluorescence polarization in the absence of immunosuppressant (B0) and the red bars correspond to the signal in the presence of 250 ng/mL MPA (B250). Results are shown as mean signals ± the standard error of the mean (n = 3).

Figure 3. Evaluation of the binding avidity and specificity of MPA-AO (1 nM) for anti-MPA, anti-tacrolimus and anticyclosporine antibodies (25 µg/mL) in PBST. The dashed line is the mean mP signal (n = 12) for MPA-AO in the absence of any antibody. Results are shown as the mean signal ± the standard error of the mean (n = 3).

Binding of MPA-AO to the antibody, in the absence of analyte, was evaluated at different anti-MPA (0.005 – 25 µg/mL) and MPA-AO concentrations (1 – 1000 nM). As shown in Figure S5 (Supporting Information), the best results were obtained using 1 nM of MPA-AO and 5 µg/mL anti-MPA antibody concentration, since such combination provides good sensitivity and precision as well as a wider dynamic range.

Optimization of the incubation time. The incubation time has usually a direct effect on the analytical characteristics of an immunoassay. The antibody-antigen binding rate in a FPIA is not limited by the diffusion of the target compound to a surface as in heterogeneous immunoassays. Therefore, the incubation time can be relatively short and must be optimized.35 The binding kinetics plots (Figure 5) were generated by recording, every 10 min, the fluorescence polarization after the addition of 5 µg/mL of anti-MPA antibody to a mixture of 1 nM MPA-AO and MPA, at concentrations in the 0 – 10 µg/mL range. The plots revealed that equilibrium was achieved after just 10 min; however, assay sensitivity (measured by its IC50) and signal reproducibility significantly improved after 20 min incubation therefore this time was selected for further assays.

Effect of the buffer composition. The effect of the buffer composition on the analytical signal was evaluated by performing the FPIA in phosphate buffer (10 mM, pH 7.4) with Tween 20 (0.05%), carbonate buffer (10 mM, pH 9.0) with Tween 20 (0.05%), PFBB, and MES buffer (10 mM, pH 5.7) with Tween 20 (0.05%). The fluorescence polarization was measured in the absence (B0) and in the presence (B250) of 250 ng/mL MPA. The data shown in Figure 4 demonstrate that PBST provided the best results, in terms of sensitivity (lowest B/B0 ratio), reproducibility and dynamic range span (Figure S6, Supporting Information).

Figure 5. Kinetics of binding of MPA for the FPIA. The black symbols correspond to the fluorescence polarization obtained in the absence of MPA and red opened symbols correspond to the



IC50 values obtained at each assayed time (MPA range: 0 – 10 µg/mL). Results are shown as mean signals ± the standard error of the mean (n = 3).

Analytical characterization. The normalized competition curve obtained with MPA standards in PBST at concentrations ranging from 0 to 10 µg/mL is depicted in Figure 6 (blue symbols). The IC50 value of the immunoassay was 7.7 ± 0.5 ng/mL and the LOD was determined to be 0.8 ng/mL. The dynamic range, calculated from 20–80% inhibition, ranged between 1.7 ± 0.2 and 39 ± 4 ng/mL. The reproducibility of the FPIA was demonstrated by relative standard deviations (RSD) < 11% for intraday assays (n = 3), and < 16% for interday assays (n = 3), for immunosuppressant concentrations in the range of 0.025 ng/mL to 10 µg/mL. Compared to previously reported immunoassays and commercial kits for MPA quantification, the developed fluorescence polarization immunoassay for free MPA analysis shows superior performance in terms of both sensitivity and reproducibility (Table S1, Supporting Information).

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Figure S7 (Supporting Information) shows the comparison of the dose-response curves obtained in either non-diluted ultrafiltered serum and with 1/4 (v/v) dilution in PBST. No statistically significant differences (P > 0.05) were observed between the calibration plots obtained in PBST or in plasma or serum diluted 1/4 (v/v) with the buffer. Thus, such dilution was used for further measurements.

Recovery studies in spiked serum and blood samples. The FP immunoassay was applied to the analysis of free MPA in commercial males serum samples spiked with the immunosuppressant at three concentration levels (1000, 5000 and 10000 ng/mL). The samples were ultrafiltered prior to analysis and the results were compared with those obtained by RRLC-DAD. As shown in Table 1, no significant differences were found between the amount of free MPA calculated by either method at any of the MPA concentration levels tested. The results suggest that, upon spiking, the major fraction of the immunosuppressant binds to the serum proteins, which are removed in the ultrafiltration step. The calculated amount of free MPA was in the range of 0.75 – 1.3%, which is in agreement with the values reported the literature for free MPA in blood samples.4,6,7,14,37 Table 1. Comparison of the performance of FPIA and RRLC-DAD for quantification of MPA in serum (n = 3) Measured concentration (ng/mL)

Free fraction (%)

MPA nominal concentration (ng/mL)

FPIA

1000

13

18

22

12

1.3

1.8

5000

41

43

5

6

0.83

0.86

10000

75

72

4

6

0.75

0.72

RSD (%)

RRLCRRLCRRLCFPIA FPIA DAD DAD DAD

RSD: Relative standard deviation

Application to the analysis of plasma samples from a transplanted patient. Finally, the suitability of the FPIA Figure 6. Dose-response plots for MPA (blue ▲, n = 9, 11 points), MPAG (green ♦, n = 3, 8 points), Tacrolimus (black ■, n = 3, 8 points) and cyclosporin A (red ●, n = 3, 8 points), using AO-labeled MPA with anti-MPA antibody in PBST. Results are shown as mean signals ± the standard error of the mean (n = 3).

The immunoassay cross-reactivity was tested with CsA and FK506, typically administered to patients in combination with MPA, and with the most common MPA metabolite, MPAG. As observed in Figure 6, CsA and MPAG induce a weak response at high concentration levels; however, crossreactivities were negligible at the concentrations found in blood (therapeutic concentration of CsA in whole blood is: 150 – 350 ng/mL).36 These results confirm the excellent specificity of the FPIA.

Matrix effect in biological samples. Biological samples could inhibit the antibody-antigen binding efficiency, or even increase the non-specific binding of MPA-AO. In order to investigate the potential existence of matrix effects, a commercial males´ serum sample and plasma from a healthy woman volunteer, not treated with any immunosuppressant, were ultrafiltered to remove the protein, and then spiked with increasing concentrations of MPA, ranging from 0 to 10 µg/mL, before the analysis.

for free MPA quantification in a plasma sample of a transplanted volunteer (treated with mycophenolate mofetil and FK506) was evaluated. After a previous ultrafiltration step to remove proteins, the sample was spiked with 36 or 72 ng/mL of MPA. As shown in Table S2 (Supplemental Information), mean recoveries were in the range of 96–103% with RSDs between 7–14%, demonstrating the applicability of the FPIA method to the analysis of free MPA in real samples. Moreover, these results were statistically comparable to those obtained by RRLC-DAD (P > 0.05).

CONCLUSIONS The results obtained in this work demonstrate the suitability of long-wavelength fluorescent labels for the successful development of screening methods based on FPIA. The latter are able to analyze immunosuppressants in blood samples in an ultrasensitive and fast manner. The FPIA method developed here allows quantification of the MPA free fraction in serum or plasma samples with a significant improvement in the LODs compared to reported immunoassays for the same purpose. Moreover, the method also shows superior performance in terms of reproducibility, and selectivity. No matrix effects


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Analytical Chemistry have been observed when the ultrafiltered serum or plasma samples were diluted (1/4, v/v) in PBST and 96 samples were analyzed in 20 min with the aid of a microplate fluorometer. The results of the analysis of free MPA in serum or plasma samples confirmed the low concentration of the free drug, which is the pharmacologically active form, present in the samples. The fluorescence polarization immunoassay reported here shows great promise for its implementation in point of care testing (POCT) devices.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Details about the synthesis and characterization of the MPA-AO. Optimization of the Amicon® ultrafiltration and pretreatment of the 96-well plate. Description of the four-parameter sigmoidal logistic equation. Saturation binding analysis. Optimization of the tracer and antibody dilutions. Effect of the buffer solution, comparative summary of analytical methods reported for the analysis of MPA in biological samples and evaluation of the matrix effect in biological samples (PDF).

AUTHOR INFORMATION Corresponding Author * Tel: (+34) 91 394 5147, fax: (+34) 91 394 4329. E-mail: [email protected] (M.C. Moreno-Bondi) and [email protected] (E. Benito-Peña)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the EU (FP7-NMP-2010-LARGE-4 contract no. 318372 “NANODEM”) and the MINECO/FEDER (CTQ2015-69278-C2) grants. B. Glahn-Martínez thanks MINECO for a Grant (“Promoción de Empleo Joven e Implantación de la Garantía Juvenil en I+D+i”). We thank M. V. Cañamares and P. Martínez for helpful discussions.

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