1. Introduction
Fetal disorders of the heart rhythm and conduction occur with a frequency from 0.1 to 5% from all pregnancies and may cause low cardiac output, fetal hydrops, intrauterine growth restriction, premature labor, and fetal, neonatal, and infant mortality [
1,
2]. The most common fetal arrhythmias (FA), >160–180 beats/min, include premature atrial contractions (PACs), the supraventricular tachycardia (SVT), and the atrial flutter (AFL). SVT and AFL are complicated by nonimmune fetal hydrops in 30–50% of FA pregnancies resulting in the perinatal mortality increase of up to 17–43% [
3,
4,
5].
The timely initiation of ART promotes the conversion of fetal tachyarrhythmia to the sinus rhythm, the regression of the concomitant non-immune fetal hydrops, and the cardiac remodeling indicators, thus decreasing the rate of perinatal morbidity and mortality. Transplacental ART contributes to the prolongation of pregnancy to full term with an average delivery at 39.8 ± 1.2 weeks [
6]. Transplacental medical interventions, including digoxin, flecainide, sotalol, and amiodarone, started more than 40 years ago. Still, there is no consensus on the first-line treatment of FA, especially SVT. Different medical centers present the significant variability in the evaluation of drug-induced rhythm recovery: from 1–5 days (M = 1 day) [
7], 1–35 days (M = 7.5 days) [
8], and up to 12 days [
9]. There are significant differences in the frequency of fetal tachycardia conversion to sinus rhythm complication with non-immune fetal hydrops (38–86%) and without hydrops (27.8–96%) [
3,
7,
8,
9]. The need of optimal treatment regimen search for the fetus with tachyarrhythmia is of high importance.
Digoxin, cardiac glycoside, is often chosen as the first-line agent for the treatment of fetal SVT without fetal hydrops. It is relatively safe and has a long history of use [
10]. The initial dosage of digoxin is 0.5–2.0 mg/day with subsequent correction [
11,
12]. Digoxin monotherapy showed a lower effective rate (27.8%) than combined with flecainide/sotalol (72.2%) [
11]. The transplacental transfer of digoxin is reduced in fetal hydrops [
13,
14]. The drug bioavailability at absorption from the gastrointestinal tract is 70–80%. Digoxin is metabolized in the liver and excreted mainly by the kidneys (50–70% is unchanged) [
12,
15].
Sotalol, a non-selective β-blocker, is used both as monotherapy and in combination with digoxin/flecainide in fetal arrhythmia [
5,
7]. The initial dosage is 160–320 mg divided b.i.d., in the absence of medical cardioversion—up to 480 mg/day [
7,
11]. The oral bioavailability of sotalol is 89–100%. The drug is not metabolized by the liver and is excreted in 80–90% unchanged by the kidneys. According to Van der Heijden et al. the use of sotalol contributed to rhythm recovery in 78% of fetuses with SVT [
6]. In the absence of a therapeutic effect within 5–8 days, the addition of digoxin or flecainide led to a successful conversion of the arrhythmia in 100% of cases [
6,
8].
Pharmacogenetic differences due to different levels of expression and activity of enzymes responsible for the acetylation, hydrolysis, oxidation, or metabolism of drugs shows a wide ethnic variation [
16]. The patients can be divided in two groups: rapidly and slowly metabolizing drugs. The
ABCB1 (
MDR1, multidrug-resistance gene) gene is located at the 7q21 locus and consists of 28 exons. The clinical significance was found for three
ABCB1 single nucleotide polymorphic (SNP) loci: 1236C > T (rs1128503), 3435C > T (rs1045642), and 2677G > T/A (rs2032582). These SNPs change the level of P-glycoprotein expression and the excretion of a number of drugs, in particular digoxin [
16]. P-glycoprotein (Pgp), encoded by the
ABCB1 gene, is an ATP-dependent membrane transporter, exporting some drugs and xenobiotics from the inside of endothelial cells to the outside. Pgp protects the cells and organs against from toxic xenobiotic agents and some drugs. It is expressed in the liver, small and large intestines, kidneys, and placenta [
17,
18].
ABCB1 3435C > T polymorphism change the level of Pgp expression [
19]. The patients with the 3435TT genotype have a low expression of the P-glycoprotein in the intestine, liver, kidneys, and placenta. Medium therapeutic doses of drugs result in higher concentrations of drugs in the blood due to an increase in bioavailability (more complete absorption by small and large intestine enterocytes, decrease in the barrier function of the placenta, and inhibition of excretion by the kidneys and liver) [
20].
The frequency of 3435C > T polymorphism of the
ABCB1 gene in the Russian Federation is 30.0% (24.8–35.6) for the TT genotype, 48.6% (42.7–54.5) for the CT genotype, and 21.4% (16.8–26.6) for CC [
21,
22]. Every third patient has a risk to develop side effects; every fifth patient metabolizes drugs quickly and most likely does not receive the expected therapeutic effect. An individual approach (low therapeutic doses) for patients with the 3435TT
ABCB1 genotype is preferable. Digoxin, a first-line ART for fetal heart rhythm disorders, with a very narrow recommended therapeutic range, is a substrate for P-glycoprotein.
ABCB1 genotyping can individualize the choice of the digoxin dosing regimen.
Digoxin and sotalol are drugs with a delayed onset of equilibrium concentration. With long-term use, a linear increase in the level of drugs is noted. The widely used and recommended therapeutic concentration range of digoxin for adults is very narrow (0.8–2 ng/mL) [
23] and, recently, it was proposed to limit this range up to 0.5–0.9 ng/mL [
24]. Controversial data on fetal/maternal serum digoxin ration were presented—from 40% up to 90% [
11]; in particular, Takekazu Miyoshi et al. showed 0 0.53 (0–1.0) value [
25] and, according to Ebara H. et al., it was 0.84 (0.6–1.0) [
26]. The ratio of the fetus to maternal blood level for sotalol was found at 1.11 (0.67–2.87) by Oudijk M.A. et al. [
27] and 1.07 (47.2–371.6) by Takekazu Miyoshi et al. [
25]. The relatively high ratio of sotalol in the amniotic fluid to the fetus blood, 3.2 (1.28–5.8), could be explained by the active renal excretion of the drug, which is not metabolized by liver [
27]. The digoxin ratio in the amniotic fluid to the fetus blood was found at 6.0 (4.0–8.0), according to Miyoshi [
25].
The accurate and precise monitoring of the antiarithmic drugs concentrations (for instance, digoxin) remains of utmost importance. In general, immunoassays are routinely employed for digoxin monitoring in mother’s blood [
28], but these techniques have relatively low specificity toward glycosides (i.e., digoxin) with high levels of cross-reactions with digoxin-like immunoreactive factors [
26,
29,
30,
31,
32]. In modern laboratories, an increasing number of studies are carried out using the gold standard for diagnostics, namely high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). There are several reports concerning the application of this analytical approach for the determination of digoxin in adults and newborns [
33,
34,
35,
36,
37,
38,
39].
There are few HPLC-MS/MS studies of digoxin and sotalol pharmacokinetics for adults and neonates. The transplacental pharmacokinetics and pharmacodynamics of common tachyarrhythmia drugs differ significantly from those for adults due to the presence of three blood circulations (maternal, fetal, and placental) and the complexity of their interaction [
26,
27,
40,
41,
42,
43,
44,
45]. It is necessary to choose the personal dosing regimen to avoid the mother/fetal intoxication or the lack of the antiarrhythmic effect [
46,
47]. The cancellation of drugs for transplacental therapy may be due to the fetus state and the development of side effects in a pregnant woman. The adverse effects occurred with a frequency of 30–38.8% and included mild headache, moderate nausea, diarrhea, and may be reduced or eliminated by reducing the dose [
23,
48]. There is a genetic predisposition to the antiarrhythmic drugs variations in pharmacokinetics, pharmacodynamics, and side effects [
20,
21,
40,
49,
50,
51]. Of particular interest is the influence of the xenobiotic metabolization gene,
ABCB1, polymorphisms to the transplacental ART efficacy.
The aim of this study was to study the pharmacokinetics of transplacental antiarrhythmic drugs using HPLC-MS/MS, as well as to assess the effect of the ABCB1 3435C > T polymorphism on the efficacy and maternal/fetal complications of digoxin treatment.
3. Discussion
The distribution of the fetal arrhythmia types and the average time for diagnosing (30.4 ± 3.8 weeks) in this study was consistent with the data available: PAC was presented in 29.2% and SVT and AFL in 70.2% of FAs [
3,
5,
9,
52,
53,
54,
55,
56,
57]. A case series of viral disease during pregnancy and subsequent fetal heart rhythm disturbance described by Hoi-Shan Chan et al., 2001, Savarese et al., 2008, Takahashi et al., 2011, and Dejog et al., 2015 attracted attention to the infectious status of a pregnant woman [
58,
59,
60,
61,
62,
63]. More frequent acute respiratory viral infections (
p = 0.025), asymptomatic bacteriuria (
p = 0.053), and impaired vaginal microflora (
p = 0.002) at the II trimester of pregnancy was found in the FA group compared to the control.
The complete medical rhythm conversion was achieved in 34.6–71.4% of cases and was significantly more often in SVT and AFL groups (
p = 0.027), which is comparable to the data from other studies (27.8–96% and 38–86% in the absence and presence of non-immune fetal hydrops, accordingly) [
3,
5,
7,
9]. The frequency of rhythm disruption on drug dosage reduction was more frequent in the fetal SVT group (
p = 0.015). It confirms the difficulties in individual selection of the initial transplacental ART and the drug dosage reduction regimen [
3,
27,
64]. The side effects requiring discontinuation and/or reduction in the dosage were 1.6 times less common than published by Moatassim et al., 2018 and Chimenea et al., 2021 (23.3% vs. 30–38.8%) [
23,
48]. These side effects (dyspepsia, digestive symptoms, and headache) were eliminated with a decrease in the dosage of the drug.
The polymorphisms of genes coding the transporters involved in detoxification changes the individual features of the drug pharmacokinetics [
17,
18,
21,
50]. The data obtained confirmed the expediency of genotyping a pregnant woman for the 3435C > T polymorphism of the
ABCB1 xenobiotic detoxification gene. The predisposition to a decrease in the drug bioavailability in patients with a homozygous 3435CC variant showed a probable association with the development of ART side effects. Dynamic electrocardiography showed a pronounced decrease in “heart rate” in the women with the 3435TT allele of the
ABCB1 gene. This can be explained by the lower expression of the ABCB1/P-glycoprotein in the intestine, liver, kidneys, and placenta. When prescribing medium therapeutic doses of P-glycoprotein substrates (namely, digoxin), a higher concentration of the drug in blood is observed due to the more complete absorption by enterocytes of the small and large intestines and the inhibition of excretion by the kidneys and liver [
17,
18,
19].
The expediency of fetal 3435C > T genotyping of the ABCB1 gene in fetal arrhythmias had also been shown. The homozygous 3435TT variant in the fetus showed a probable association with an earlier response to ART and rhythm disruptions on the digoxin dosage reduction. The confirmation of the ABCB1 3435C > T polymorphism influence on the course of fetal arrhythmia requires a multicenter, prospective, randomized, and controlled trial.
The features of drug pharmacokinetics during pregnancy are due to physiological changes in the mother’s body and the presence of an additional fetoplacental circulation affecting the distribution, metabolism, and elimination of the drug [
41,
42,
65]. The fetal invasive examination is limited. A non-invasive method to determine the antiarrhythmic drug concentration in the fetal blood based on pregnant women plasma or urine drug levels possesses a high value.
The enzyme-linked immunosorbent assay (ELISA) remains the most common method for digoxin measurement. This analytical method has several limitations, including the low specificity and insufficient limit of quantification. The high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) is the “gold standard” for therapeutic drug monitoring. The advantages of the HPLC-MS/MS over the enzyme immunoassay method are high accuracy, sensitivity, specificity, and ability to measure drugs in various types of the biological samples (blood, urine, amniotic fluid, etc.). HPLC-MS/MS guarantees the optimal sensitivity to clarify the transplacental drugs pharmacokinetics [
66] in the case of low concentrations (ng/mL for digoxin).
The HPLC–MS/MS method developed in this study was used to measure digoxin and sotalol blood plasma, amniotic fluid, and urine with adequate sensitivity and selectivity, high accuracy and precision, and a low matrix effect [
38,
66,
67,
68,
69,
70,
71,
72]. The high selectivity was obtained by the usage of three MRM transitions for each compound studied (analyte and IS). Similar to most of the previously published HPLC-MS/MS methods, digitoxin,
Digitalis glycoside, was used as an internal standard for digoxin [
38,
66,
67,
68,
69,
70,
71,
72]. The digoxin LOQ was 0.2 ng/mL, which is lower than the widely used therapeutic range for adults (0.8–2 ng/mL). A low digoxin level, 0.42 (0.20, 0.66) ng/mL, was found in the cord blood of newborns with transplacental ART at delivery.
The results obtained in the study of the pharmacokinetics of antiarrhythmic drugs showed a high degree of correlation between the level of digoxin and sotalol in maternal and cord blood. It allowed for fetal therapeutic drug monitoring using the mother plasma HPLC-MS/MS-MRM analysis. The “therapeutic window” achievement for digoxin in the blood of a pregnant woman expects a proportional level in the fetal blood plasma. The ratio of digoxin in cord blood to maternal blood, 0.35 (0.26:0.46), was lower than previously published by Takekazu Miyoshi et al. (0.53 (0 and 1.0)) [
25] and Ebara H. et al. (0.84 (0.6 and 1.0)) [
65]. These differences may be explained by drug withdrawal before delivery and the drug metabolism peculiarities under the multidrug-resistance gene polymorphisms. More accurate regression between fetus and maternal digoxin in blood was calculated after excluding babies with fast drug metabolisms (3435CC genotype of
ABCB1 gene). The fetus/maternal digoxin ratio for TC and TT babies was 0.38 (0.26 and 0.45) (r
S = 0.87).
The level of digoxin in the blood of the fetus at the first rhythm recovery episode was lower (0.58 (0.46 and 0.80) ng/mL) than the widely used and recommended therapeutic range for adults (0.8–2.0 ng/mL). This may be the reason for the need of the second antiarrhythmic drug (sotalol).
The digoxin ratio in the amniotic fluid to the fetus blood was 3.37 (2.23 and 5.93) at the 8–10 day of ART and 10 (7.87 and 14.9) at delivery, which corresponds to the multicenter trial by Takekazu Miyoshi (6.0 (4.0–8.0)). The highest concentration of digoxin in the amniotic fluid was found in the fetal TC and TT genotype compared to the CC genotype (p = 0.031). These differences may be explained by the slower elimination of the antiarrhythmic drug in fetuses with the 3435CC ABCB1 variant.
The sotalol ratio in fetus cord blood to maternal plasma was 1.0 (0.97:1.07), which is consistent with data from other studies: 1.11 (0.67–2.87) by Oudijk M.A. et al. [
27] and 1.07 (47.2–371.6) by Takekazu Miyoshi et al. [
25]. It should be expected that there are almost identical sotalol levels in the blood of mothers and the fetuses after achieving equilibrium. The ratio of the sotalol in amniotic fluid to fetal plasma was 4.88 (4.84 and 9.29) at delivery and 3.75 (1.68 and 5.77) at the 6th (4 and 8) day of ART, similar to Oudijk M.A.’s study at 3.2 (1.28–5.8) [
27]. The pronounced accumulation of sotalol in the amniotic fluid may be the result of the effective renal excretion of the unchanged drug. This study confirmed the high efficacy of the placental transfer of sotalol (almost 100%) and the absence of individual patient characteristics’ (including the
ABCB1 genotype) effect on the pharmacokinetics of the drug. Thus, sotalol ART is more predictable for the mother and fetus. Several studies have shown the advantage of sotalol as a first-line drug in fetal antiarrhythmic therapy [
13,
27]. Sotalol, a non-selective β-blocker, prevents high blood pressure in pregnant women also [
73].
The efficacy of the drug transplacental transfer depends on the placental perfusion, the pH values of the maternal and fetal blood, and the physicochemical properties of the drug (size, protein binding, hydrophobicity, etc.). The level of most drugs “behind the placenta” (fetal blood and amniotic fluid) varies from 20 to 80% of the level in the mother’s blood [
26,
27]. The immaturity of the fetal kidneys leads to the elimination of drugs by back diffusion through the placenta into the mother’s bloodstream. Part of the drug metabolites become more polar, impairing transplacental excretion, and accumulating in various tissues of the fetus. The maturation of the fetus urinary system increases the excretion of drugs into the amniotic fluid. The fetus swallows the drug and its active metabolites from the amniotic fluid, thus increasing the effect of the drug [
26,
27].
The pharmacogenetic differences in the xenobiotic detoxification gene (
ACBC1) varies significantly [
20,
41,
46,
74]. Single nucleotide polymorphisms divide patients into rapidly and slowly metabolizing medicines [
75]. An individual approach in the dosing of ABCB1/glycoprotein-P substrates is recommended, in particular, to reduce the dose of digoxin [
46,
76,
77] for the 3435TT ABCB1 variant. The success of transplacental antiarrhythmic therapy largely depends on the drug dosage penetrating the placenta to the fetus. P-gp minimizes the transplacental transfer of substrates, enhancing the barrier function of the placenta [
78]. The significant decrease in placental P-gp expression with advancing gestation results in the increase in the digoxin effect on the fetus in late gestation [
17]. The down regulation or inhibition of P-gp expression in fetuses with 3435CC
ABCB1 polymorphism may improve the access of digoxin to the fetus and enhance the antiarrhythmic effect.
This study had several limitations. First, this was a single center prospective study. The main advantage of prospective data and sample collection was a single treatment protocol. However, the fetal ART was not randomized between several institutions. This was the largest prospective study in the limited number of cases per year (89 participants) [
57]. However, therapeutic drug monitoring of digoxin/sotalol (HPLC-MS/MS) and ABCB1 gene polymorphism detection was performed for a limited number of patients (30 mother-child pairs). Thirdly, only 3435C > T polymorphism of the multidrug-resistance gene was studied in this work. Other polymorphisms of
ABCB1 and genes responsible for the acetylation, hydrolysis, oxidation, or metabolism of drugs may also influence the pharmacokinetics and pharmacodynamics of antiarrhythmic drugs. Further multicenter studies are needed to establish the most effective approaches to fetal ART.
4. Material and Methods
4.1. Study Design
The clinical part of the work was carried out by the 1st Department of Obstetric Pathology of Pregnancy of the National Medical Research Center for Obstetrics Gynecology and Perinatology named after the academician V.I. Kulakov of the Ministry of Healthcare of the Russian Federation. The samples were collected from October 2018 to January 2021. The study included 89 pregnant women with a prenatally diagnosed fetal heart rhythm disorder of the tachyarrhythmia type and ART and 50 healthy pregnant women (the control group). All the patients signed a voluntary informed consent to participate in the study. This work was approved by the Ethical Committee of the National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov (protocol No. 9, dated 22 November 2018).
The fetal arrhythmia was established on the basis of a change in the frequency and/or regularity of the fetal “heart rate” during auscultation or ultrasound examination and confirmed by echocardiographic examination. The criteria for inclusion were the fetal arrhythmias diagnoses established using ultrasound examination and confirmed with echocardiography, singleton pregnancy, informed agreement to participate in the study, and fetal antiarrhythmic drugs medication. The patients with multiple pregnancies, chromosomal abnormalities in the fetus, and maternal diseases excluding the use of the fetal therapy were not included. The exclusion criteria were the lack of desire/ability to continue the participation in the study as well as the serious side effects of ongoing therapy.
All the patients underwent laboratory (blood electrolytes, thyroid hormones, and glycated hemoglobin levels) and instrumental (electrocardiography, expert ultrasound examination of the fetus, Doppler ultrasonography of the vessels of the uteroplacental and fetal-placental blood flows, cardiotocography, and echocardiographic examination of the fetus) studies. In case of fetal tachyarrhythmia, a diagnostic transabdominal amniocentesis was performed under ultrasound-assistance in order to detect an infectious (viral and bacterial) etiological factor in the development of the pathology.
All the patients had different dosages of antiarrhythmic drugs during treatment (
Supplementary File S2). The starting dose of digoxin for all the patients was 0.75 mg/day and decreased with the development of side effects and/or according to the results of routine therapeutic monitoring of digoxin. The starting dose of sotalol was 160 mg/day and, in the absence of a therapeutic effect, increased to 480 mg/day. The daily dosages of drugs were provided in
Supplementary File S2 for each sample collection time coinciding with the patient’s visit to the doctor. During the time interval between samplings (
Table 6), the dosage did not change. The digoxin medication time was at 07.00/15.00/23.00 and sotalol at 08.00/20.00. The maximum concentration of digoxin/sotalol was achieved in 1–2 h and 2–3 h, respectively. For the simultaneous measurement of these drugs, the sampling was performed three hours after the last medication, at 10.00.
The HPLC- MS/MS-MRM studies of plasma blood, amniotic fluid, and cord plasma blood were performed four times during fetal ART for 30 pregnant women and newborns to assess the transplacental pharmacokinetics of sotalol and digoxin.
Table 6 illustrated the type of samples at four time points collected for this group. The dates of sample collection were available in
Supplementary File S2. For these 30 pregnant women and 18 newborns, the 3435C > T polymorphism of the ABCB1 gene was screened also. The number of patients who underwent therapeutic drug monitoring decreased from 89 to 30 due to missing several visits to the doctor (the absence of samples at more than one time point), patient refusal of amniocentesis, or delivery in another medical center (especially in preterm birth). The first sample was collected at the 3rd (2 and 5) day, the second at 6th (4 and 8) days, and the third at the 9th (9 and 10) day.
The following HPLC-MS/MS investigations were performed for each pair of mother/fetus: 4 tests of the pregnancy blood, 4 tests of the pregnancy urine, 1 test of the newborn blood, and 2 tests of the amniotic fluid. The sum amounts of the samples were 99 maternal plasma blood, 97 maternal urine, 40 samples of the amniotic fluid, and 19 samples of the plasma cord blood.
4.2. Sample Collection
The amniotic fluids samples were obtained during transabdominal amniocentesis under ultrasound control and at delivery. All the surgical interventions were performed by one team of surgeons. The amniotic fluid was collected in 15 mL plastic tubes followed by centrifugation (10 min at 12,000 rpm at 40 °C). The supernatant was collected in 2 mL cryotubes (1.7–2 mL each).
The maternal venous blood was extracted into a 3 mL EDTA tube followed by centrifugation at 3000 rpm in 20 min at 40 °C, the supernatant was centrifugated at room temperature in 10 min at 12,000 rpm and collected in 2 mL cryotubes (0.5–1 mL each). The newborn cord blood was extracted into a 1 mL EDTA tube. The sample preparation procedure was the same as the maternal blood.
The average portion of urine (not the morning one) was collected under aseptic conditions in a plastic container (15 mL) followed by centrifugation (10 min at 12,000 rpm at 40 °C) and the supernatant were extracted into 2 mL cryotubes (1.7 mL each).
The processing and storage were carried out within 30 min after extracting the biomaterial. The labeled tubes were frozen at −80 °C and stored until the measurements.
4.3. Sample Preparation for HPLC-MS/MS
The measurements of the concentration of the antiarrhythmic drug (digoxin and sotalol) in the plasma of a pregnant woman and a newborn, urine, and amniotic fluid of a pregnant woman were performed by HPLC (Agilent, Santa Clara, CA, USA) with tandem mass-spectroscopy (SCIEX, Framingham, MA, USA).
The MTBE (≥99.5%) HPLC grade was obtained from Fisher Chemical (Loughborough, UK). The methanol (MeOH) (99.9%) HPLC grade was obtained from Scharlab S.L. (Barcelona, Spain). The ultrapure deionized water was obtained with a Milli-q reference water purification system (Millipore Corporation, Billerica, MA, USA). The acetonitrile (99.9%) HPLC grade was obtained from Fisher Chemical (Loughborough, UK). The formic acid (98%), ammonium acetate (98%), sotalol hydrochloride analytical standard (308.82 g/mol), digoxin analytical standard (780.94 g/mol), digitoxin analytical standard (764.94 g/mol), and atenolol analytical standard (266.34 g/mol) were obtained from Merck KGaA (Darmstadt, Germany). All the stock solutions were prepared by dissolving the required amount of the soltalol/atenolol/digoxin/digitoxin in 100% MeOH. The solutions were stored in glass vials at −80 °C in a refrigerator prior to use.
The sotalol sample preparation procedure was the following: 100 µL of a sample and 10 µL of the internal standard (atenolol) were mixed; the 1800 µL of ACN:MeOH = 3:1 mixture was added; and, after 5 min centrifugation, 1000 µL of the supernatant was transferred into a vial for HPLC-MS/MS analysis. The digoxin sample preparation procedure included the addition of 20 µL IS (digitoxin) to 400 µL; 1 mL MTBE addition to the mixture and centrifugation for 5 min; drying of 850 µl of the supernatant in the N2 stream at room temperature; and precipitate dilution in 100 mL of ACN:H2O = 1:1 mixture followed by centrifugation for 5 min with subsequent transfer to a vial with an insert for HPLC-MS/MS analysis. The plasma blood of the healthy volunteer was used as a matrix for the plasma blood HPLC-MS/MS analysis. The urine of the healthy volunteer was used as a matrix for the urine and amniotic fluid analysis.
4.4. HPLC-MS/MS Parameters
The chromatographic separation was carried out in an HPLC system Agilent 1260 infinity (Agilent, Santa Clara, CA, USA) consisting of a degasser, a pump, an autosampler, and the column with a thermostat. The mass spectrometer was calibrated according to standard ABSciex procedure using PPG Positive solution (2 × 10−7 M) from an ABSciex calibration kit. The solution was injected at 5 µL/min. The list of the monitored masses included m/z 59.050, 175.133, 500.380, 616.464, and 906.673. The tuning was performed until the mass error becomes less than 0.1 and peak width—between 0.6–0.8. The procedure was repeated for both quadrupole 1 and 3 at scan rates of 10, 200, 1000, and 2000 Da/s.
A Waters SPHERISORB column (Waters, Milford, Massachusetts, USA) 2.1 × 150 mm × 5 µm without a guard column was used for sotalol measurement. Ultrapure H2O with the ammonium acetate (10 mmol/L) was used as a mobile phase A. Undiluted acetonitrile (100%) was used as a mobile phase B. The measurements were performed in isocratic mode during 5 min at a constant phase ratio A/B 40/60. The flow was 800 µL/min. The injection volume was 5 µL. The column temperature was 40 °C. The system dead time was less than 30 s under these conditions and the retention time was 2.3 min and 2.7 min for sotalol and IS, respectively.
A Poroshel 120 EC-C18 column (Agilent, Santa Clara, CA, USA) 2.1 × 150 mm × 2.7 µm was used for the digoxin analysis with a guard column Zorbax (Agilent, Santa Clara, CA, USA) 2.1 × 5 mm × 1.9 µm. Ultrapure H
2O with formic 0.1% acid and the ammonium acetate (10 mmol/L) was used as a mobile phase A. An ACN:H
2O = 9:1 mixture with 0.1% formic acid and ammonium acetate (10 mmol/L) was used as a mobile phase B. The measurements were performed during 7 min: the first 1.5 min the ratio A/B was 9:1, after it initially increased to 5/95 and maintained a constant during 3 min then instantly returned to the initial parameters and maintained a constant during 2.5 min. The flow rate was 500 µL/min. The injection volume was 25 µL. The column temperature was 25 °C. The system dead time was less than 30 s and the retention time was 3.3 min and 3.6 min for digoxin and IS, respectively.
Table 3 illustrated the multiple reaction monitoring (MRM)-selected transitions of sotalol, digoxin, and its internal standards.
4.5. HPLC-MS/MS MRM Methods Validation
The HPLC-MS/MS MRM methods were validated per the ICH M10 bioanalytical method validation guidelines [
79], including sensitivity (LLOD and LLOQ), selectivity, linearity, intra- and interday precision, matrix effect, recovery, accuracy, and robustness. The blank matrixes of plasma blood, urine, and amniotic fluid experiments were created as a mixture of 10 heathy volunteers’ blood and urine.
The calibration curve for digoxin in amniotic fluid (
Figure S1, Supplementary File S1) was based on nine points in the range from 0.5–10 ng/mL with four levels of quality control (QC). The calibration levels were 0.5, 0.6, 1, 1.5, 2.5, 5, 6, 7.5, and 10 ng/mL. The QC levels were 0.5, 1.5, 5, and 7.5 ng/mL. The chromatograms for the blank and the samples are illustrated in
Figure S2, Supplementary File S1.
The calibration curve for digoxin in blood plasma (
Figure S3, Supplementary File S1) was based on nine points in the range from 0.2–10 ng/mL with four levels of QC. The calibration levels were 0.2, 0.5, 1, 1.5, 2.5, 5, 6, 7.5, and 10 ng/mL. The QC levels were 0.6, 1.8, 5, and 7.5 ng/mL. The chromatograms for the blank and the samples are illustrated in
Figure S4, Supplementary File S1.
The calibration curve for digoxin in urine (
Figure S5, Supplementary File S1) was based on seven points in the range from 10–1000 ng/mL with four levels of QC. The calibration levels were 10, 25, 50, 100, 200, 500, and 1000 ng/mL. The QC levels were 10, 25, 200, and 500 ng/mL. The chromatograms for the blank and samples are illustrated in
Figure S6, Supplementary File S1.
The calibration curve for sotalol in amniotic fluid (
Figure S7, Supplementary File S1) was based on nine points in the range from 0.2–10 ng/mL with four levels of QC. The calibration levels were 0.2, 0.4, 0.5, 0.6, 1, 2, 4, 8, and 10 ng/mL. The QC levels were 0.2, 0.6, 5, and 7.5 ng/mL. The chromatograms for the blank and the samples are illustrated in
Figure S8, Supplementary File S1.
The calibration curve for sotalol in blood (
Figure S9, Supplementary File S1) was based on nine points in the range from 0.2–10 µg/mL with four levels of QC. The QC levels were 0.2, 0.6, 5, and 7.5 µg/mL. The chromatograms for the blank and the samples are illustrated in
Figure S10.
All the calibration curves were created using linear weighted (1/×2) regression analysis of the normalized peak areas (drug / IS area) with a correlation coefficient above 0.99.
To evaluate the selectivity of 10 healthy volunteers’ human plasma and urine, the spiked LLOQs and high QCs were tested. A low limit of detection (LLOD) was found as the digoxin or sotalol concentration at the S/N ratio of more than three. The lower limit of quantitation (LLOQ) was calculated as the lowest amount of analyte, quantitatively determined with acceptable (<20%) precision and accuracy. To study intra- and interday precision and accuracy five replicates of four QCs (LLOQ, low QC, medium QC, and high QC) for digoxin and sotalol were analyzed at three different days. The accuracy of the method was found as intra- and interday percent deviation (DEV) from the QC concentration nominal value (
Table S2, Supplementary File S1). The intraday precision was described in terms of √ (within-day mean square)/GM × 100%, the interday precision √ (between-day mean square/n)/GM × 100%, where GM (grand mean) was the mean of the observed concentrations across run days using one-way analysis.
The matrix effect (%) and extraction recovery (%) were evaluated three times using the blank sample (ultrapure H
2O with LLOQ, medium and high QC digoxin/sotalol, and IS) and the blank matrix spiked before and after the extraction (
Table S2, Supplementary File S1). To estimate the robustness, we slightly modulated the HPLC conditions, such as the flow rate, mobile phase (ammonium acetate in phase A), and column temperature at about 10% range. The validation data for each analyte and matrix were summarized in
Table S4, Supplementary File S1.
4.6. ABCB1 Gene Polymorphism Study
The
ABCB1 gene single nucleotide polymorphism (SNP) study of 3435C > T locus was carried out for 30 pregnant women and 18 newborns using a polymerase chain reaction (PCR) with a consequent readout of melting curves using a modified kissing probes (adjacent probes) assay [
80]. The venous blood cell fraction (erythrocytes and leukocytes) was aliquoted in cryotubes and stored at −80 °C. The DNA extraction was performed using a Prep-GS-Genetics kit (DNA-Technology JSC, Moscow, Russia) according to instructions. The DNA concentration was measured on a DNA minifluorimeter (Hoefer, USA). The typical values were in the range from 50 to 100 µg/mL. The genotyping was carried out using the original labeled oligonucleotides for the modified kissing probe assay (DNA-Technology JSC, Moscow, Russia). The DNA amplification, fluorescence, and melting temperature measurements were performed on the DT-96 Real-Time PCR Cycler (DNA-Technology JSC).
4.7. Statistical Analysis
The statistical data processing was performed in the RStudio (1.383 GNU) using our own scripts in the R language (4.1.1). The Shapiro–Wilk test was used to check the normality of the data distribution. Median values (Me) and quartiles (Q1, Q3) were used to describe the quantitative data without normal distribution. The qualitative data were presented as absolute values (%). The comparative analysis for qualitative data was performed using a Fisher’s exact test, χ2-test. The comparative analysis of the quantitative data was carried out using the Mann–Whitney test for the pairwise comparison of groups and the Kruskal–Wallis test for the comparison of more than two groups. The significance threshold was determined to be 0.05.
The correlation coefficients between two quantitative variables were estimated using Spearman’s rank correlation. The modeling using the Passing-Bablok linear regression method was applied to develop a predictive model for a quantitative variable from another quantitative variable.