Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435

Home , Ambrisentan

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Journal of Pharmaceutical and Biomedical Analysis

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Bionalytical validation study for the determination of unbound

ambrisentan in human plasma using rapid equilibrium dialysis

followed by ultra performance liquid chromatography coupled to

mass spectrometry

a a a,∗ a

Soledad Garcia-Martínez , Estitxu Rico , Enriqueta Casal , Alba Grisalena˜ ,

a a a b a

Eider Alcaraz , Nicholas King , Nerea Leal , Iker Navarro , Miguel Ángel Campanero

Bioanalytical services, Dynakin, S.L, Bizkaia Technology Park, 48160 Derio, Bizkaia, Spain

Noorik Biopharmaceuticals, Lange Gasse 15, 4052 Basel, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Ambrisentan is a highly selective endothelin-1 type A receptor antagonist indicated for use in the

Received 10 July 2017

treatment of pulmonary hypertension. In this study an assay was developed and validated for the quan-

Received in revised form

tiﬁcation of total and unbound (free) concentrations of ambrisentan in human plasma. Plasma samples

13 December 2017

were dialysed against phosphate buffered saline in a rapid equilibrium dialysis device to obtain dialysate

Accepted 15 December 2017

and plasma for unbound and total ambrisentan, respectively. Subsequently, ambrisentan and deuterated

Available online 16 December 2017

ambrisentan (internal standard) were extracted from plasma or plasma dialysate by solid-phase extrac-

tion and separated by ultra performance liquid chromatography using on a reversed-phase C column.

Keywords: 18

Detection was conducted with a tandem mass spectrometer with an electrospray ionization source and

Unbound ambrisentan

analysed in positive ion mode with multiple reaction monitoring. Calibration curves were generated over

Total ambrisentan

Rapid equilibrium dialysis (RED) a linear concentration range of 0.1–200 ng/mL in plasma and 0.1–10 ng/mL in plasma ultraﬁltrate; with a

Ultra performance liquid recovery for ambrisentan of 69.4% and 77.5%, respectively. This assay has been shown to be reproducible

chromatography–tandem mass and sensitive. The lower limit of quantiﬁcation in both cases was 0.1 ng/mL; reaching a sensitivity not

spectrometry (UPLC–MS/MS)

previously described in the literature. The inter- and intra-batch precision and accuracy were in both

cases ≤±15%. The procedure was applied to assess total and free plasma concentrations of ambrisentan

in healthy volunteers. Plasma protein binding of ambrisentan was approximately 99%.

1. Introduction AMB is highly protein-bound (98.8%), and is eliminated predom-

inantly by nonrenal mechanisms [2,3]. Therefore, the dose of AMB

Endothelin-1 is a potent vasoconstrictor and is mediated by two does not need adjustment in individuals with mild to moderate

types of endothelin receptors including type A and type B. Over- renal impairment. There are currently no data on the use of AMB in

expression of endothelin-1 has been demonstrated in patients with patients with severe renal impairment.

pulmonary arterial hypertension [1]. Ambrisentan (AMB), a highly Plasma protein binding of a drug can vary in various pathological

selective endothelin-1 type A receptor antagonist, is currently and physiological states, acting as a potential source of variability

approved in the United States of America, Europe and different in clinical efﬁcacy. Hepatic impairment and nephritic syndrome are

countries for the treatment of this vascular pathology. AMB blocks associated with hypoalbuminemia which could potentially lead to

the effects of endothelin-1 and thus decreases blood pressure in altered protein binding levels of a drug.

the lungs. The thickening of blood vessels in the lungs and heart is Determination of plasma protein binding of drugs is critical in

also inhibited by AMB. the drug development since only the free drug concentration is

considered as the active fraction, because it is able to pass through

membranes to reach tissues and penetrate into cells to exert its

effect. Plasma protein binding of a drug is clinically important, since

it has profound effects on both the pharmacokinetics and pharma-

∗

Corresponding author. codynamics, as well as the safety margin [4,5]. The study of free drug

E-mail address: [email protected] (E. Casal).

https://doi.org/10.1016/j.jpba.2017.12.030

428 S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435

concentrations can be of great utility whenever plasma protein used to prepare quality control (QC) samples, calibration standards,

binding changes occur, improving the therapeutic drug monitor- zeros and blank samples in plasma and plasma ultraﬁltrate, respec-

ing approach based on total drug concentrations and contributing tively. Individual human plasma and plasma ultraﬁltrate requested

to a better individualization of drug dosage regimen. Therefore, for the assessments of selectivity and matrix effect were provided

determination of unbound AMB concentrations in plasma may be by Sera-Lab Ltd. and BBI Solutions (Le Perray, France). Human whole

helpful in better characterizing the pharmacokinetics of the AMB blood was collected (by Laboratorio Axpe, Spain) from healthy

in a variety of clinical settings. donors, into collection tubes pre-treated with K2-EDTA.

The study of unbound concentration requires two steps. Firstly

the separation of the unbound and bound fractions. There are a 2.2. Preparation of calibration standards, quality control samples

number of commonly used methods available for the separation and internal standard solutions

of the unbound and bound fractions; these include ultraﬁltra-

tion, ultracentrifugation, gel ﬁltration, capillary electrophoresis A stock solution of 1.0 mg/mL AMB in dimity lsulphoxide was

and equilibrium dialysis. Extensive reviews of all the techniques used for preparation of calibration standards and QC samples. The

can be found in the literature [6,7]. However, rapid equilibrium solubility of AMB in different solvents was initially evaluated (data

dialysis (RED) has long been considered the gold standard method not shown). The results demonstrated that the AMB is soluble in

since non-speciﬁc adsorption of drugs to the device and membrane dimetyl sulphoxide, acetonitrile and acetonitrile:water (1:1, v:v)

has less impact than other techniques [8–10]. In addition, is the and slightly soluble in water and 0.1% formic acid in acetoni-

most amendable to automation, because it can be implemented trile:water (2:8, v:v). The stock (1 mg/mL in dimetyl sulphoxide)

◦

using simple liquid handling techniques. was stored protected from the light at −20 C and it was stable for

The second step in the determination of unbound concentra- at least eighteen days.

tion of a drug requires a methodology to separate and quantify the Two sets of intermediate calibration solutions were prepared by

drug; usually via chromatographic techniques coupled to different diluting of the stock solution in acetonitrile:water (1:1, v:v). One

types of detectors. A thorough literature survey has revealed that set (10000, 9000, 2500, 250, 50, 25, 10 and 5 ng/mL) was prepared

the methods currently available for analysis of AMB include ultra- for determination of total AMB in plasma. Another set (500, 450,

violet in pharmaceuticals and related substances [11–13] or mass 250, 200, 50, 25, 10 and 5 ng/mL) was prepared for determination

spectrometry detection in biological samples or pharmaceutical of AMB in plasma ultraﬁltrate. These intermediate calibration solu-

◦

degraded samples [14–18]. None of these methods have been used tions were stored protected from the light at −20 C and these were

to quantify the unbound AMB fraction. In addition, these methods stable for at least seven days.

are not sufﬁciently sensitivity to measure the concentration of the The intermediate calibration solutions were used to prepare cal-

unbound fraction. ibration curves in plasma at concentrations of 200, 180, 50, 5, 1, 0.5,

The aim of this study was to develop a RED based assay 0.2 and 0.1 ng/mL and in plasma ultraﬁltrate at concentrations of

followed by a solid phase extraction (SPE) ultra performance liq- 10, 9, 5, 4, 1, 0.5, 0.2 and 0.1 ng/mL. The calibration curves were

uid chromatography tandem mass spectrometry (UPLC–MS/MS) freshly prepared on the day of sample processing.

to measure the unbound AMB fraction in human plasma. This Two sets of intermediate QC solutions (stable for at least seven

◦

methodology required to perform the validation of two indepen- days at −20 C) were prepared by diluting of the stock solution

dent methods: (1) determination of total AMB concentration in (1 mg/mL) in acetonitrile: water (1:1, v:v). One set (160 ␮g/mL and

human plasma and (2) determination of unbound AMB concentra- 10000, 8000, 500, 15 and 5 ng/mL) was prepared to evaluate total

tion in human plasma dialysate. Both quantiﬁcation methods were AMB in plasma. Another set (8000, 500, 400, 100, 15 and 5 ng/mL)

based on the same SPE and UPLC–MS/MS method. was prepared to evaluate AMB in plasma ultraﬁltrate. These sets

In this paper we describe a validated bioanalytical assay for were subsequently used to prepare QC samples (dilution quality

the quantiﬁcation of AMB in plasma and dialysate. In addition, we control, upper limit of quantiﬁcation (ULOQ), high quality control

present data from a pharmacokinetics study in which AMB was (HQC), medium quality control (MQC), low quality control (LQC)

administrated to healthy volunteers via intravenous infusion. This and lower limit of quantiﬁcation (LLOQ)) in plasma at concen-

proposed new route of administration for AMB could be useful in trations of 3200, 200, 160, 10, 0.3 and 0.1 ng/mL and in plasma

situations in which the normal oral administration route is not ultraﬁltrate at concentrations of 160, 10, 8, 2, 0.3 and 0.1 ng/mL,

appropriate. respectively. QC samples were stored in propylene tubes and frozen

◦

at -80 C until use, except those used for to evaluate the long term

◦

stability at −20 C. These QC samples were analysed following one

2. Experimental freeze thaw cycle to represent an analytical sample undergoing

initial analysis. The dilution QC samples were diluted with blank

2.1. Chemicals and reagents matrix before processing (factor dilution 1 in 20 for plasma and 1

in 10 for plasma ultraﬁltrate).

AMB, chemically known as (2S)-2-[(4,6-dimethylpyrimidin- For the IS, a 1.0 mg/mL stock solution of [ H10]-AMB was pre-

2-yl)oxy]-3-methoxy-3,3-diphenyl-propanoic acid (>99% purity), pared in dimetyl sulphoxide and stored protected from the light

◦

was provided by TLC Pharmachem (Vaughan, Canada) and at −20 C; this solution was stable for at least forty days. An inter-

the internal standard (IS) [ H10]-ambrisentan (>99% purity) by mediate working solution (1 ␮g/mL) of IS was prepared by further

Alsachim (Strasbourg, France). Dimetyl sulphoxide was obtained dilution with acetonitrile:water (1:1, v:v); this solution was stable

◦

from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline at −20 C for at least seven days. A working solution of IS (1 ng/mL)

(dialysis buffer containing 0.1 M sodium phosphate and 0.15 M in 2% formic acid was prepared just prior to use and was not stored.

sodium chloride; pH 7.2), methanol, acetonitrile and formic acid This solution was used for the plasma assay and for the dialysate

were provided by Thermo Fisher Scientiﬁc (Waltham, MA, USA). assay.

All mobile phase solvents were LC/MS grade. Milli-Q water was

obtained in-house using a Millipore system (Bedford, MA, USA). 2.3. Extraction in plasma and plasma ultraﬁltrate or dialysed

Two pools of drug-free (blank) human plasma and blank human

plasma ultraﬁltrate, with K2-EDTA as the anticoagulant, were pur- For determination of total and unbound AMB fraction, 100 ␮L

chased from Sera-Lab Ltd. (Sussex, England). These pools were of plasma or plasma ultraﬁltrate/dialysate (calibration standards,

S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435 429

QC samples, zeros, blank samples or real samples) were mixed ultraﬁltrate sample processed without AMB and but with IS) and

for 10 min with 400 ␮L IS solution (1 ng/mL) in the wells of a 96- at least one “blank” sample (plasma or plasma ultraﬁltrate sample

deep-well polypropylene plate. After the addition of the IS, plasma processed without AMB and without IS) after the highest calibra-

samples were processed by SPE using Oasis HLB 96-well plates tion sample. In the case of “accurate and precision” batches six

(30 mg sorbent per well, 30 ␮m particle size; Waters Corp., Mil- replicates of each QC sample (LLOQ, LQC, MQC, HQC and ULOQ)

ford, MA, USA). The extraction plates were ﬁrst conditioned with were included.

200 ␮L of methanol and then equilibrated with 200 ␮L of water.

␮

Subsequently, the diluted samples with the IS (500 L) were loaded 2.5.1. Selectivity

␮

to the SPE wells. The SPE wells were then washed with 200 L of The selectivity in both methods (plasma or plasma ultraﬁltrate)

5% methanol in water and ﬁnally each well was eluted under vac- was determined using drug-free plasma or plasma ultraﬁltrate

␮

uum with 100 L of methanol and the eluents were collected into from six different individuals (including four non-haemolysed and

a clean 96-well collection plate. The wells of the collection plate two haemolysed samples). Haemolysed samples were prepared by

◦

were subsequently dried under nitrogen ﬂow at 40 C. The result- adding haemolysed whole blood to the blank samples to produce

␮

ing residues were reconstituted each with 50 L of 0.1% formic acid a 2% haemolysed whole blood solution. The selectivity was tested

␮

in acetonitrile:water (2:8, v:v) and mixed for 10 min 5 L of sample both with and without the addition of IS. The selectivity samples

were injected onto the UPLC–MS/MS system. were extracted as described in Section 2.3 and analysed using the

method described in Section 2.4. The selectivity was evaluated by

2.4. UPLC–MS/MS conditions comparing the area quantiﬁed on the area of the chromatogram

corresponding to the analyte and IS in the selectivity samples to

Chromatographic separation was carried out using an Agilent the mean area of the LLOQ samples (six replicates).

(Palo Alto, CA, USA) UPLC system consisting of an Agilent 1290

binary pump, an Agilent 1290 inﬁnity standard autosampler and 2.5.2. Matrix effect on ionization

an Agilent 1290 inﬁnity thermostated column compartment. The The effect of matrix was evaluated for AMB in plasma and plasma

◦ ◦

cooled autosampler was set to 4 C and the column oven to 30 C. ultraﬁltrate from six different individuals. Each set of samples

× ␮

A Kinetex EVO C18 column (50 2.1 mm, 1.7 m) provided by Phe- included: non-haemolysed blank samples (four plasma samples)

nomenex (Torrance, USA) was used for separation of AMB. A binary and haemolysed blank samples (two samples). For each method,

gradient method was developed using solvent A (0.1% formic acid two sets of these blank matrix samples were processed as described

in water) and solvent B (0.1% formic acid in acetonitrile). The gra- in Section 2.3 and the ﬁnal extracts, before injecting into the UPLC

dient was as follows: from 80 to 5% A in 3.5 min, constant at 5% system, were spiked with AMB and IS equivalent in concentra-

A for 0.5 min, back to 80% A in 0.1 min, and constant at 80% A for tion to that which would have be found in a sample assuming

0.9 min. The ﬂow rate was 0.25 mL/min. The injection volume was 100% recovery in the ﬁnal extract. One set of blank matrix was

␮

set at 5 L. reconstituted with the low QC spiking solution. Another set was

The UPLC system was coupled to an Agilent 6495 triple reconstituted with a high QC spiking solution. Both spiking solu-

quadrupole mass spectrometer for the detection of the AMB and tions were prepared in 0.1% formic acid in acetonitrile:water (2:8,

internal standard [ H10]-AMB. The mass spectrometer was oper- v:v). To determine matrix effect, peaks areas of blank samples

ated in electrospray positive mode (ESI + ). Data was acquired using spiked with the spiking solutions were compared to peak areas of

the following settings: capillary voltage 6000 V, drying gas tem- six spiked solutions (without extracted matrix) at corresponding

◦

perature 125 C, gas ﬂow 20 L/h, nebulizer pressure 60 psi, sheath concentrations in solution (0.1% formic acid in acetonitrile:water

◦

gas temperature 300 C, sheath gas ﬂow 8 L/h, high pressure ion (2:8, v:v), representing 100% (no matrix effect).

funnel RF voltage 100 V and low pressure ion funnel RF voltage The matrix factor (MF) was used to assess relative matrix effects.

60 V. Detection of AMB and [ H10]-AMB was achieved by multiple

reaction monitoring (MRM) at m/z transitions of 379.1 > 303.2 and

Area of analyte in the presence of matrix

389.2 > 313.2, respectively. The dwell time was set to 200 ms, the MF

Mean area of analyte in the spiking solution

fragmentator 380 V and the collision energy to 8 V.

Agilent MassHunter data acquisition for triple quadruple mass

Area of IS in the presence of matrix

spectrometer (version B 07.01) was used for data acquisition and ISmatrixfactor =

Mean area of IS in the spiking solution

Agilent MassHunter quantitative software (version B 07.01) was

used for data processing.

Analyte matrix factor

IS normalized matrix factor =

IS matrix factor

2.5. Speciﬁc assay validation

To quantify the unbound AMB fraction in human plasma was 2.5.3. Recovery

necessary to validate two independent bioanalytical methods. One The recovery of AMB in plasma and plasma ultraﬁltrate was

a method for the determination of total AMB in plasma and the evaluated by processing LQC, MQC and HQC samples (six replicates

other a method to quantify AMB in dialysed plasma. Both meth- of each) in the normal manner. The peak areas obtained from these

ods were validated according to the FDA and EMA guidelines for samples were then compared with the peak areas obtained from

validation of bioanalytical assays [19,20] for linearity, sensitivity, processed blank matrix samples that had been spiked post extrac-

accuracy and precision, selectivity, matrix effect, recovery, dilution tion to equivalent levels assuming 100% recovery (six replicates per

integrity and stability. Both methods were validated by analysis level).

of QC samples prepared as previously described and included in

an analytical batch. Each analytical batch was performed on a 96- 2.5.4. The stability of AMB in different conditions

well plate and included: conditioning samples (at least eight), a 2.5.4.1. Whole blood stability. Whole blood (using K2-EDTA as the

calibration curve (in the range of 0.1–200 ng/mL for plasma or anticoagulant) from an individual human was spiked with AMB

0.1–10 ng/mL for plasma ultraﬁltrate), one set of duplicate QC sam- (100 ng/mL). Aliquots of the whole blood were centrifuged after

◦

ples in plasma or plasma ultraﬁltrate at three concentration levels 0, 30, 60, 90 and 120 min post equilibration (15 min at 37 C) to

(LQC, MQC and HQC), at least one “zero” sample (plasma or plasma produce plasma, which was then frozen prior to analysis. This was

430 S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435

Table 1

were extracted (Section 2.3) and ﬁnally analysed by UPLC–MS/MS

Flow rages of ambrisentan for groups A and B.

(Section 2.4).

Time (hours) Group A Flow Group B Flow RED was conducted to obtain the unbound fraction of AMB in

rate (␮g/h) rate (␮g/h)

plasma samples. The single-use plate RED inserts contained dialy-

0–4 75 300 sis membrane with a molecular weight cut-off of approximately

4–12 44 200 8000 Da (ThermoFisher Scientiﬁc, Waltham, MA, USA). The pro-

12–16 100 500

cedure for RED was as follows. Aliquots of plasma (300 ␮L) and

16–24 75 75

dialysis buffer (buffer solution similar to plasma) (500 ␮L) were

added to sample chamber and buffer chamber, respectively on the

RED device. Using the appropriate amount of buffer is essential to

performed at room temperature. The derived plasma samples were

maintain the liquid level in both chambers. The loaded dialysis plate

analysed in triplicate and the different time points were compared.

was sealed and placed on a shaker at approximately 250 rpm and

◦

◦ ◦ incubated at physiological temperature (37 C) for 4 h. Both the vol-

−

2.5.4.2. Bench top, long term stability at 20 C and −80 C and freeze

ume ratio and the incubation conditions established in the dialysis

thaw stability. The stability of AMB in human K2-EDTA plasma or

process were performed according to the manufacturer of the RED

plasma ultraﬁltrate was investigated under different conditions. ◦ ◦

device. The dialysate was stored at −80 C ± 10 C prior to assay.

Two levels of QC samples (LQC and HQC; six replicates per level)

The dialysate samples (100 ␮L) were quantiﬁed in a batch contain-

prepared in matrix were used to evaluate the stability of AMB under

ing: calibration samples (in the range of 0.1–200 ng/mL), duplicate

different conditions. The conditions evaluated were: (1) stability at

◦ ◦ QC samples at three concentration levels (0.3, 10 and 160 ng/mL),

room temperature for 24 h, (2) stability at −20 C ± 5 C for 35 days,

◦ ◦ zero samples and a blank sample. Then, the samples were extracted

(3) stability at −80 C ± 10 C for 35 days and (4) stability following

and analysed by UPLC–MS/MS in the same manner as the plasma

four freeze thaw cycles. In this case, the QC samples were frozen at

− ◦ ◦ samples.

80 C ± 10 C for a minimum of 12 h and then defrosted at room

In this study, incurred sample reanalysis (ISR) was performed

temperature for 2 h.

on 40 samples for the total concentration of AMB and 16 samples

for the free concentration. The samples were selected using the

2.5.4.3. Processed sample integrity. The stability of processed sam-

following criteria: (1) samples were above LLOQ, (2) samples cov-

ples (prepared using both methods; plasma and ultraﬁltrate) was

ered the whole range of doses included in the study, (3) samples

assessed by the injection of an “accuracy and precision” batch after

◦ were reanalysed within the stability period and (4) samples were

5 days storage at nominally 10 C.

from the maximum plasma concentration and from the elimination

phase.

2.5.4.4. Re-injection stability. The stability of AMB in re-injected

samples was assessed by the re-injection of a previously accepted

“accuracy and precision” batch following three days storage under

3. Results and discussion

the same conditions as expected in the autosampler (nominal ◦

10 C). 2

3.1. Characterization of ambrisentan and [ H10]-ambrisentan by

UPLC–MS/MS

2.6. Application of the assay

The chromatographic conditions described in this analytical

In this study, the objective was to characterise the pharmacoki- procedure were achieved after investigating different columns and

netic proﬁle of total and unbound AMB in healthy volunteers when several mobile phases. Symmetrical and sharp peak for AMB was

administered AMB via intravenous. The AMB was administered observed for using an EVO C18 column and a mixture of acetonitrile

over a 24 h period using two different ﬂow rates proﬁles. A total −water (in both cases with formic acid 0.1%) as mobile phase. Under

of 20 volunteers were included in 2 study groups (group A: n = 10, chromatography conditions the retention times of AMB and the IS

total dose = 1.652 mg of AMB; group B: n = 10, total dose = 7.800 mg). were 2.8 min (Fig. 1). The total run time was only 5 min. According

Both groups were infused with a 5 mg/mL solution but the ﬂow rate to the full-scan ESI (+) mass spectra, the protonated molecule ion

+ + 2

was altered to change the infusion rate. The infusion proﬁles for [M + H] m/z 379.1 for AMB and [M + H] m/z 389.2 for [ H10]-AMB

groups A and B are presented in Table 1. were selected as the precursor ions to obtain the product ions. The

The study complied with the recommendations of the 18th most sensitive mass transition from the precursor ions to the prod-

World Health Congress (Helsinki) [21] and all applicable amend- uct ion were m/z 379.1 → 303.2 and m/z 389.2 → 313.2. AMB in the

ments. The study also complied with the international and local ESI + mode shows a selective loss of the CH3OH followed by CO2,

applicable laws, regulations and guidelines and was approved by resulting in the product ion m/z 303 [14] (see Fig. 1).

the corresponding regional ethics committee.

Blood samples, using K2-EDTA as the anticoagulant, were taken

from the volunteers at the following time points: pre-dose (–1 h), 2, 3.2. Validation

4, 6, 9, 12, 14, 16, 18, 21, 24, 26, 28, 32, 36, 42, 48 and 72 h post-dose

for the quantiﬁcation of total AMB. The unbound plasma concen- In this study, we develop a RED assay followed by SPE and

trations of AMB were determined at the following time points: UPLC–MS/MS to measure the unbound AMB fraction in human

pre-dose (–1 h), 4, 12, 16, 24 and 36 h post-dose. Following the plasma. This methodology required the validation of two inde-

collection, whole blood samples were centrifuged (at 2000g for pendent methods: (1) determination of total AMB concentration

ten min) and, once centrifuged, all plasma samples were frozen in human plasma and (2) determination of unbound AMB concen-

◦ ◦

at −80 C ± 10 C prior to assay. tration in human plasma ultraﬁltrate. The Table 2 summarizes the

Initially, the determination of the total concentration of AMB results of both methods.

was carried out. Plasma samples (100 ␮L) were included in a batch Acceptance criteria were established for the overall analytical

containing: calibration samples (in the range of 0.1–200 ng/mL), run. For the calibration samples were that the back-calculated con-

duplicate QC samples at three concentration levels (0.3, 10 and centrations of 75% (8 out of 10) standards should fall within ±15%

160 ng/mL), zero samples and a blank sample. Then, the samples (± 0% at the LLOQ) of the nominal value. Values falling outside these

S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435 431

Fig. 1. Representative multiple reaction monitoring chromatograms of ambrisentan (m/z 379 > 303) and its internal standard (m/z 389 > 313) at the lower limit of quantiﬁcation

(LLOQ; 0.1 ng/mL) in human plasma (A) and plasma ultraﬁltrate (B). Representative chromatograms of blank plasma (C) and plasma ultraﬁltrate (D) without internal standard.

The proposed pattern of fragmentation for ambrisentan is shown on top.

limits were excluded. In addition the coefﬁcient of determination 3.2.1. Evaluation of the linearity of the calibration curve

(r ) of the calibration curve should be >0.9800. Calibration curves (in the range 0.1–200 ng/mL for plasma and

Acceptance criteria for the QC samples were that the measured 0.1–10 ng/mL for plasma ultraﬁltrate) were prepared in blank

concentrations of at least 67% (4 out of 6) of the QC samples should matrix for each batch (a total seven calibration curves by each

fall within ±15% of the nominal value. Two out of six QC sam- method). All the calibration curves in this study were freshly pre-

ples could fall outside this limit as long as they were both not at pared on the day of sample processing. Each batch contained at

the same nominal concentration. Additionally, acceptance crite- least one zero sample and at least one blank sample. These zero

ria for the individual assessments were also established. These are and blank samples were used for evaluating possible interferences,

described in detail in each Section. without including them in the calibration curve. The calibration

models were assessed to be linear regression with 1/␹ weighting

432 S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435

Table 2

the previously ﬁxed (1.0 ng/mL, [15]) in biological samples with a

Summary of method validation data for the measurement of total ambrisentan (in

similar run time.

plasma) and free ambrisentan (in plasma ultraﬁltrate).

The ULOQ or the highest concentration that can be measured

Species Human

with acceptable precision and accuracy were also evaluated. The

Matrix K2-EDTA plasma and plasma

ULOQ of these assays was 200 ng/mL for plasma and 10 ng/mL for

ultraﬁltrate

plasma ultraﬁltrate. In both methods, acceptable values of precision

Analyte Ambrisentan

≤ ±

Internal standard [ H10]-ambrisentan (CV% 15%) and accuracy (bias within 15%) at the ULOQ were

Calibration range 0.1–200 ng/mL (plasma) and 0.1– demonstrated (Table 3).

10 ng/mL (plasma ultraﬁltrate)

The carryover was assessed by injecting four blank plasma sam-

Lower limit of quantiﬁcation 0.1 ng/mL

ples after the injection of the highest calibration sample. The area

Sample volume 100 ␮L

responses in the blank samples were compared to the mean area

Method outline SPE followed by detection with

UPLC–MS/MS response of the LLOQ QCs in the same batch. No carryover was

Calibration model Linear regression

detected for the AMB or IS in plasma and plasma ultraﬁltrate, with

Weighting factor 1/␹

no peaks found with response >20% of the corresponding mean

Within batch accuracy and Requirements fulﬁlled

precision response of the analyte in the LLOQ QCs, and no peaks found with

Between batch accuracy and Requirements fulﬁlled response >5% of the corresponding mean response of the IS in the

precision LLOQ QCs.

Selectivity Negligible

Matrix effect on ionization Negligible

3.2.3. Selectivity

Recovery ambrisentan 69.4% (plasma) 77.5% (plasma

ultraﬁltrate) The selectivity in both methods (plasma or ultraﬁltrate) was

Carryover Negligible

evaluated by comparing chromatograms of blank samples and

Dilution integrity Determinations after 20 fold dilution

plasma or plasma ultraﬁltrate spiked with AMB at LLOQ level. No

(in plasma) and 10 (ultraﬁltrate)

◦ ◦ endogenous components interfering with the detection of AMB

Stock solution stability At least 18 days at −20 C ± 5 C

(1 mg/mL) were found in the chromatograms of blank samples. Representative

◦ ◦

− ±

Working solution stability At least 7 days at 20 C 5 C MRM chromatograms of blank plasma and ultraﬁltrate without the

◦ ◦

Isotopic purity of the internal At least 40 days at −20 C ± 5 C

addition of the IS are included in Fig. 1C and D, respectively.

standard (1 mg/mL)

◦ ◦

Internal standard working At least 7 days at −20 C ± 5 C

solution (1 ␮g/mL) stability 3.2.4. Matrix effect on ionization

Long term stability at At least 35 days

The determination of ionization suppression (or enhancement)

− ◦ ◦

80 C ± 10 C

by matrix extracts is important as this phenomenon can severely

Long term stability at At least 35 days

◦ ◦ affect the sensitivity and robustness of the analytical method. Sam-

−20 C ± 5 C

Processed samples integrity At least 5 days ples of matrix from different sources were analysed. The matrix

Re-injection stability At least 3 days effect was evaluated by comparing the peak area ratio in the post-

Freeze thaw stability At least 4 cycles

extracted plasma samples at two concentration levels (LQC and

Bench top stability At least 24 h at room temperature

HQC) with neat standard solutions at the same concentration levels.

Whole Blood Stability At least 120 min at room temperature

The ion suppression or enhancement of the signal in terms of IS-

normalized matrix factor was found to be at LQC level 0.954 ± 3.0%

2 and 0.992 ± 0.6% and at HQC level 0.919 ± 4.7% and 0.988 ± 0.6%

and the r for each curve were greater than 0.9800. For both meth-

for AMB in plasma and plasma ultraﬁltrate, respectively. Therefore,

ods, the results from the calibration standards were found to be

acceptable precision (CV of IS-normalized matrix factor ≤15%) was

precise (coefﬁcient of variation or CV ≤ 6.8%) and acceptable (bias

demonstrated for both methods and the results indicated that any

within ±4.0%).

matrix effect on the ionization of AMB was consistent and did not

compromise the quantiﬁcation of AMB in plasma or ultraﬁltrate

3.2.2. Accuracy and precision, sensitivity, lower and upper limit

plasma.

of quantiﬁcation and carryover

A total of three “accuracy and precision” batches for method

3.2.5. Recovery

were processed. Each “accuracy and precision” batch included: con-

The overall recovery for AMB and the IS was calculated by com-

ditioning samples, a calibration curve in matrix, at least one zero

paring the peak-area ratios of spiked samples before and after the

sample, four blank samples after the highest calibration sample and

extraction in different lots of plasma and plasma ultraﬁltrate at

six replicates of each QC sample (LLOQ, LQC, MQC, HQC and ULOQ)

three QC levels (LQC, MQC, and HQC). The mean recovery of AMB

prepared in matrix. The results obtained for these QC samples were

was 69.4% and the IS normalised recovery had a CV% of 5.6%.

used to calculate the within and between-batch accuracy and pre-

In plasma ultraﬁltrate the mean recovery of AMB was high

cision. Acceptable accuracy (bias within ±15% or ±20% at the LLOQ)

(77.5%) and consistent (CV% of 4.8%) and the recovery of the IS was

and precision (CV ≤ 15% or ≤20% at the LLOQ) for the determination

similar to AMB.

of total AMB in plasma and AMB in dialysate plasma were demon-

strated. The results from the within and between-batch accuracy

3.2.6. Dilution integrity

and precision measurements are reported Table 3.

The samples were analysed and the results (corrected for dilu-

These “accuracy and precision” batches were also used to eval-

tion) compared with the nominal concentration. The ability to

uate the limits of quantiﬁcation of both methods. The sensitivity

perform accurate determinations after a 20-fold dilution for total

or the lowest concentration of a sample (LLOQ) that can be quan-

AMB in plasma and 10-fold dilution for AMB in plasma ultraﬁltrate

tiﬁed with acceptable precision and accuracy were evaluated in

were demonstrated after dilution with blank matrix of six repli-

both methods. Acceptable precision (CV ≤ 20%) and accuracy (bias

cates of a dilution QC sample (3200 ng/mL in plasma and 160 ng/mL

within ±20%) at the LLOQ was demonstrated for AMB in plasma

in plasma ultraﬁltrate). The results obtained were precise (3.6%

and for AMB in plasma ultraﬁltrate (Table 3). Representative chro-

and 6.7%) and accurate (6% and -4% bias) for plasma and plasma

matograms from the LLOQ level in plasma and ultraﬁltrate plasma

ultraﬁltrate, respectively.

are shown in Fig. 1. The achieve LLOQ (0.1 ng/mL) was lower than

S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435 433

Table 3

Within and between-batch precision and accuracy in quality control samples prepared in plasma and plasma ultraﬁltrate.

Plasma

LLOQ LQC MQC HQC ULOQ

(0.1 ng/mL) (0.3 ng/mL) (10 ng/mL) (160 ng/mL) (200 ng/mL)

◦

Batch N 1

Within-batch mean (ng/mL) 0.103 0.291 10.0 173 210

Within-batch CV (%) 15.1 4.9 1.6 11.2 1.5

−

Within-batch bias (%) 3 3 0 8 5

n 6 6 6 6 6

◦

Batch N 2

Within-batch mean (ng/mL) 0.111 0.323 10.2 168 213

Within-batch CV (%) 2.3 2.0 1.0 2.3 2.2

Within-batch bias (%) 11 8 2 5 7

n 5* 6 6 6 6

◦

Batch N 3

Within-batch mean (ng/mL) 0.100 0.283 9.69 157 209

Within-batch CV (%) 3.8 1.7 1.2 2.6 2.3

Within-batch bias (%) 0 −6 −3 −2 5

n 6 6 6 5* 6

Between-batch mean (ng/mL) 0.104 0.299 10 167 210

Between-batch CV (%) 9.9 6.7 2.3 7.8 2.1

Between-batch bias (%) 4 0 −1 4 5

n 17 18 18 17 18

Plasma Ultraﬁltrate

LLOQ LQC MQC HQC ULOQ

(0.1 ng/mL) (0.3 ng/mL) (2 ng/mL) (8 ng/mL) (10 ng/mL)

◦

Batch N 1

Within-batch mean (ng/mL) 0.0898 0.282 1.80 6.92 8.79

Within-batch CV (%) 2.1 3.8 2.3 2.7 3.7

Within-batch bias (%) −10 −6 −10 −14 −12

n 6 6 6 6 6

◦

Batch N 2

Within-batch mean (ng/mL) 0.0935 0.269 1.86 7.24 9.74

Within-batch CV (%) 0.3 0.7 4.0 1.9 9.1

Within-batch bias (%) −7 −10 −7 −10 −3

n 6 6 6 6 6

◦

Batch N 3

Within-batch mean (ng/mL) 0.0886 0.270 1.85 7.37 9.00

Within-batch CV (%) 4.7 2.3 1.0 1.5 1.8

Within-batch bias (%) −11 −10 −8 −8 −10

n 6 6 6 6 6

Between-batch mean (ng/mL) 0.0906 0.273 1.83 7.18 9.18

Between-batch CV (%) 3.6 3.3 2.9 3.3 7.3

Between-batch bias (%) −9 −9 −9 −10 −8

*A QC level was excluded according to Qtest.

3.2.7. AMB in matrix: stability determinations 3.2.7.2. Bench top stability. The stability of AMB in matrix (plasma

Drug stability in a biological ﬂuid is a function of the storage or plasma ultraﬁltrate) at room temperature for 24 h was tested

conditions, the chemical properties of the drug, the matrix, and and demonstrated using LQC and HQC samples (six replicates per

the container system. The stability of the analyte should be evalu- level). The stability of AMB under these conditions was demon-

ated during sample collection and handling, after long-term (frozen strated for both methods. The precision (CV%) was less than 3.9%

at the intended storage temperature) and short-term (bench top, and the accuracy (expressed as bias) when compared with the nom-

room temperature) storage, and after going through freeze and inal concentration was less than ±14%.

thaw cycles and the analytical process. Conditions used in stabil-

◦ ◦

− −

ity experiments should reﬂect situations likely to be encountered 3.2.7.3. Long term stability at 20 C and 80 C. The stabilities of

during actual sample handling and analysis. AMB when stored LQC and HQC (prepared with plasma or plasma

◦ ◦ ◦ ◦

ultraﬁltrate) at −20 C ± 5 C or −80 C ± 10 C were assessed for

3.2.7.1. Whole blood stability. The stability of AMB in whole blood 35 days. In both cases, the stability of AMB under these conditions

was assessed to ﬁnd possible problems during the collection and was demonstrated (% CV was less than 7.8% and the bias was less

processing of samples. Aliquots of whole blood spiked with AMB than ±11%).

were centrifuged after 0, 30, 60, 90 and 120 min after the equi-

libration period. The response of the samples left for 30, 60, 90 3.2.7.4. Freeze thaw stability. The stability of AMB in human K2-

and 120 min were within ±6% of the sample after the equilibration EDTA plasma and plasma ultraﬁltrate after four freeze thaw cycles

period. Therefore, the stability of AMB in human whole blood for was also demonstrated (% CV ranged from 1.1% to 4.1% and bias

−

120 min was demonstrated at room temperature. from 13% to 1%).

434 S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435

360 Rate 1 Rate 2 Rate 3 Rate 4 340 (4 h) (8 h) (4 h) (8 h)

320

300 Group A 280

260 Group B

240

220

200

180

160

140

120

100

0 04812 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 Time (hours)

4.0 Rate 1 Rate 2 Rate 3 Rate 4 (4 h) (8 h) (4 h) (8 h) 3.5 Group A

Group B 3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

-1.0

0510 15 20 25 30 35

Fig. 2. Mean plasma concentration–time curves of total ambrisentan (A) and unbound ambrisentan (B) in human plasma after intravenous administration (Group A :

total dose = 1.652 mg; Group B : total dose = 7.800 mg). The continuous infusion rates used in each group are included in Table 1. Each point represents the mean ± S.D.

S. Garcia-Martínez et al. / Journal of Pharmaceutical and Biomedical Analysis 150 (2018) 427–435 435

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