A sensitive method for the simultaneous UHPLC-MS/MS analysis of milrinone and dobutamine in blood plasma using NH₄F as the eluent additive and ascorbic acid as a stabilizer

Graphical abstract

Highlights

• •Simultaneous HPLC-MS/M method for quantification of milrinone and dobutamine.
• •Microanalytical method using only 20 µL of plasma and achieving LLOQ of 0.97 ng/mL.
• •Method has been used to quantify analytes from neonatal and paediatric clinical trial samples.
• •Use of NH₄F provided signal enhancement in MS detection for both analytes.
• •Dobutamine instability was improved with addition of ascorbic acid to plasma samples.

Abstract

The purpose of this work was to develop and validate an HPLC-MS/MS method suitable for quantifying two important cardiovascular drugs, milrinone and dobutamine, in neonatal and paediatric patients’ blood plasma samples. Sufficiently low LLOQ levels were required to obtain adequate pharmacokinetic data for the evaluation of optimal dosing. Since the specifics of the patient group set some restrictions on the available sample volume, the method was designed to use only 20 µL of plasma for the analysis. Analytes were separated chromatographically in a biphenyl column using a conventional water-methanol-formic acid eluent with the addition of ammonium fluoride. The latter provided a significant signal enhancement in positive ion mode detection for both analytes allowing the LLOQ to reach below 1 ng/mL. Matrix matched calibration was linear in the range of 1–300 ng/mL, between-run accuracy remained within 107–115%, and precision within 4.8–7.4% for both analytes over the calibration range (including LLOQ level). Dobutamine degradation in plasma samples was prevented by the usage of ascorbic acid. The method was applied to plasma samples of neonates from two pharmacokinetic/pharmacodynamics studies (n = 38).

1. Introduction

Milrinone’s (Fig. 1, A) primary function is to inhibit the enzyme phosphodiesterase 3 and, therefore, influence the muscle contractions in the myocardium and vascular smooth muscle. Inhibitors of phosphodiesterase reduce the breakdown of cyclic 3,5 adenosine monophosphate (cAMP) and have therapeutic influence not only on the heart but also on the lungs, where platelet functions and mechanisms control inflammatory response. Milrinone’s elimination half-life is 2.3 h, and main route of disposal is via the urine. There are two main excretion products – milrinone (83%) and its glucuronide metabolite (12%) [1]. The number of infants exposed to milrinone therapy has increased fourfold since 2005. Indications for milrinone treatment in the neonatal period include persistent pulmonary hypertension and low cardiac output syndromes, like after bypass surgery or after surgical ligation of the open arterial duct [2]. In the majority of cases dopamine is also administered, however, it is not the only concomitantly used medication, other examples include furosemide, fentanyl, midazolam and dobutamine [2].

Fig. 1.

Chemical structures of milrinone (A) and dobutamine (B).

Dobutamine (Fig. 1, B) is commonly used following heart surgery, in patients suffering a heart attack or in various states of shock. In neonates, dobutamine is the second or third most common inotrope used to treat circulatory compromise, for example, after birth asphyxia or in septic shock and to support transitional circulation in very preterm infants [3]. The commercial drug is a mixture of two optical isomers. Dobutamine increases cardiac output, lowers central venous and pulmonary artery wedge pressures and alleviates congestive heart failure symptoms, however, at high dosages, it can also lead to arrhythmia [4].

Several studies have focused on establishing dosing regimens for milrinone and dobutamine in various age groups [5], [6] and conditions. However, pharmacokinetic and pharmacodynamic data in difficult to predict populations, like neonates and especially preterm infants, are limited due to the complexity of study management [7]. In addition to ethical issues related to clinical research in minors, limited blood volume needs to be considered. According to European Medicines Agency’s guideline on the investigation of medicinal products on in term and preterm neonates, the blood loss should not exceed 3% of calculated circulating blood volume (CCBV) during the study period [8]. Heidmets et al. have shown that in very low birth weight neonates 2.3% of CCBV can be drawn for PK samples without compromising either basic hemodynamic parameters, haemoglobin values, blood component transfusions or fluid requirements [9]. Even in a sparse sampling population, PK study minimizing sample volume allows more valuable data points.

A number of methods have been developed to measure milrinone and dobutamine concentrations, however, each has its limitations, especially in cases of limited sample volume and where simultaneous measurement of multiple analytes is required. In total, 10 liquid chromatography (LC) methods were reviewed. The lowest limits of quantification (LLOQ) ranged from 0.3 to 50 ng/mL for milrinone [10], [11], [12], [13] and 1 ng/mL–27 mg/mL for dobutamine [14], [15], [16], [17], [18], [19]. The majority used conventional LC columns such as C18 [10], [11], [13], [14], [15], [16], [17], [18] and routine mobile phases, for example, ammonium formate, methanol or acetonitrile [10], [11], [12], [13], [14], [19]. Sample volume ranged from 50 to 1000 µL of plasma [11], [12], [13], [14], [15], [16], [17], [18], [19] or serum [10], depending on sample preparation method – liquid–liquid extraction [10], [16], solid phase extraction [11], [12], protein precipitation [14], [16], [19] or other [18]. Four assays used mass spectrometric (MS) detection [10], [11], [13], [19], two used UV-Vis detectors [12], [14] and those methods from the previous century used either fluorescence [15], [18] or electrochemical detection [16], [17]. Three additional methods were reviewed that had detected dobutamine hydrochloride in medicine [20], [21] and medicine or urine [22] using spectrofluorimetric methods with limits of quantification spanning 0.4–20 µg/mL [20], [21], [22]. To the best of our knowledge, no method has yet combined the simultaneous detection of both analytes in low sample volumes. However, as both drugs are often used concomitantly in clinical practice, this would allow better quantification of specific drug-related pharmacodynamic effects.

For the improvement of MS signal, especially when the sample volume is limited, novel eluent additives have been demonstrated to be beneficial [23], [24]. We have previously worked successfully with ammonium fluoride (NH₄F) and demonstrated its suitability for detection and quantification of steroid-like molecules in instances where significant signal enhancement was observed in positive ion mode [25]. Therefore, this additive was of interest to us under the scope of our current research work.

We aimed to develop a method suitable for quantifying two cardiovascular drugs, milrinone and dobutamine, under limited sample volume conditions with sufficiently low LLOQ levels for obtaining adequate pharmacokinetic data for the evaluation of optimal dosing in the future.

2. Materials and methods

The studies were approved by the Ethics Committee of the University of Tartu and registered at the EU Clinical Trials Register under numbers 2015-000486-31 and 2015-004836-36. Milrinone PK was studied in preterm neonates undergoing surgical closure of open arterial duct and at risk of post ligation cardiac syndrome, dobutamine PK and effect was studied on preterm and term neonates under various clinical statuses involving circulatory compromise within the first three days of life. For method development and validation, no clinical study samples were analysed. Blood plasma with EDTA and Citrate Phosphate Dextrose Solution (CPD) was purchased from the Blood Centre of Tartu University Hospital.

2.1. Chemicals

Standard substances for milrinone (United States Pharmacopeia Reference Standard) and dobutamine hydrochloride (United States Pharmacopeia Reference Standard) were purchased from Sigma Aldrich (Missouri, USA). Internal standard (IS) dobutamine-D4 hydrochloride was obtained from Toronto Research Chemicals (Ontario, Canada) and milrinone-D3 from TLC Pharmaceutical Standards (Ontario, Canada).

Other reagents used: HPLC grade methanol (MeOH) from Sigma Aldrich (Missouri, USA), LC-MS grade formic acid from Sigma Aldrich (Missouri, USA), LC-MS grade NH₄F from Sigma Aldrich (Missouri, USA). Reagent grade ascorbic acid was obtained from Sigma Aldrich (Missouri, USA). Water was purified (18.2 MΩ·cm at 25 °C and a total organic carbon (TOC) value 2–3 ppb) in-house using a Millipore Advantage A10 system from Millipore (Bedford, USA).

2.2. Preparation of the stock solutions, calibrators and quality control samples

Stocks at a concentration of 1 mg/mL were prepared in methanol. Sub-stocks were prepared by diluting stock solutions with water containing 1 µg/mL of ascorbic acid. For spiking calibrators and quality control samples, blank plasma was also spiked with 1 µg/ml ascorbic acid. 20 µL calibrators and quality controls were aliquoted to 200 µL PCR tubes and stored at −70 °C.

2.3. Sample preparation

For sample preparation, 20 µL of calibrators and quality control samples were thawed at room temperature. Study samples were stabilised with 1 µL of 0.2 g/mL ascorbic acid in H₂O before thawing and aliquoting 20 µL of plasma for analysis. 160 µL of 0.075% v/v formic acid in methanol and 20 µL of internal standard (IS) solution in methanol (dobutamine-D₄ and milrinone-D₃ both at a concentration of 44 ng/mL) were then added to each calibrator, quality control sample and study sample. The samples were shaken for 2 min at 2500 rpm, on an Eppendorf MixMate, and subsequently centrifuged for 5 min at 7879×g and 4 °C. The supernatant was transferred to autosampler vials and an aliquot of 2 µL was injected into the UHPLC–MS/MS system [26].

2.4. Chromatographic conditions

The Agilent 1290 Infinity (Santa Clara, USA) ultra-high performance chromatographic (UHPLC) system with a binary pump, thermostated column compartment and autosampler (thermostated at 4 °C) was used. For the chromatographic separation a Phenomenex Biphenyl analytical column (50 × 2.1 mm, 1.7 µm, 100 Å) was selected. To protect the column, an in-line filter and a Phenomenex Biphenyl guard column (10 × 2.1 mm, 1.7 µm) was installed in front of the analytical column.

The aqueous phase of the eluent comprised 0.1% formic acid and 2.5 mM NH₄F in water. For analysis, gradient elution with methanol (Table 1) was used with flow rate 0.3 mL/min. At the end of every run, the column was flushed with pure methanol.

Table 1.

Gradient program.

Time, min	Aqueous phase A, %	Organic phase (MeOH), %
0	85	15
3	70	30
4.5	0	100
5.7	0	100
6.7	85	15
10	85	15

2.5. Mass spectrometry

The Agilent 6495 Triple Quadrupole (Santa Clara, USA) served as the mass analyser. A Heated ESI ionization source (Agilent JetStream Technology) in the positive mode was used. Nitrogen served as a nebulizing, sheath, drying and collision gas. Sheath gas temperature was set to 350 °C and drying gas temperature to 260 °C. The data were collected in MRM mode; the transitions monitored, and the respective collision energies are listed in table 2. The LC-MS system was controlled with the Agilent MassHunter Workstation software version B.07.00. For peak integration and quantitative calculations, the Agilent MassHunter Quantitative Analysis software version B.07.00 was used.

Table 2.

Monitored transitions (with used collision energies, CE) and respective chemical structures of fragment ions for analytes and IS-s. Analyte transitions and structures found using online database [27].

Analyte	Precursor ion[M+H]+	Quantifier		Qualifier
	m/z	m/z	CE, V	m/z	CE,V
Milrinone	212	104 [C7H6N]+	45	194 [C12H8N3]+ 184 [C11H10N3]+ 140 [C10H6N]+ 117 [C8H7N]+	25 25 38 48
Milrinone-D3	215	142 [C9H6N2]+	44	187 [C11H10D3N3]+ 169 [C10H7N3]+ 160 [C10H9 D3N2]+ 104 [C7H6N]+	25 28 28 44
Dobutamine	302	137 [C8H9O2]+	23	166 [C10H16NO]+ 119 [C8H7O]+ 107 [C7H7O]+ 91 [C7H7]+	18 35 36 50
Dobutamine-D4	306	141 [C8H9D4O2]+	23	166 [C10H16NO]+ 158 [C8H8D4NO2]+ 123 [C8H7D4O]+ 107 [C7H7O]+	18 18 42 36

*All precursor ions were [M+H]⁺.

2.6. Calibration

A total of 8 calibrators were prepared in human plasma at concentrations of 1, 3, 8, 20, 40, 80, 160 and 300 ng/mL. Quality control samples were prepared from different stock solutions at four levels (1, 3, 70 and 220 ng/mL plasma concentration). Linear regression with 1/x² weighting was used for generating the calibration curves.

2.7. Method validation

The method was fully validated according to the European Medicines Agency’s (EMA) guideline [28]. Linearity was evaluated by comparing the experimental and measured concentrations of the calibration solutions on different days during the validation. Inter- and intra-day accuracy and precision were determined by running a batch of calibrators and quality controls on three different days; selectivity was evaluated by injecting blank plasma samples from six separate sources; carry-over was calculated from double blank plasma samples immediately following injection of the highest calibrator.

Matrix effects were evaluated as described by Matuszewski et al. [29] by comparing the post-extraction spiked plasma samples and standard solutions in the neat solvent (at the same concentration level) at two (high and low) concentrations in four replicates. Plasma for matrix effect experiments was obtained from 6 sources, additionally, hyperlipidaemic plasma and haemolysed plasma (containing 2.5, 5, 7.5 and 10% of whole blood) were tested.

2.8. Stability

During initial stability experiments, severe degradation of dobutamine was observed. To counter this, introduction of antioxidant was investigated. Stability assessment was conducted by comparing plasma with antioxidant (ascorbic acid) added before storage and/or during stability testing (stabilised plasma), to plasma without any stabilising agents (unstabilised plasma). In addition, we compared stability over time in different storage conditions. We evaluated short-term, long-term, freeze-and-thaw, and on-instrument stabilities with 6 replicates each at both low (3 × LLOQ) and high (ULOQ) concentrations. For short-term stability evaluation, the spiked samples were kept for 1, 2 and 24 h at 20 ⁰C ± 2 ⁰C, as well as for 24 h at 4 ⁰C; for on-instrument stability in the autosampler, the prepared samples were kept at 4 ⁰C for a minimum of 24 h. A total of three freeze-and-thaw cycles were applied for freeze and thaw stability and for long-term stability the spiked plasmas were stored for four months at −70 ⁰C.

2.9. Method implementation to clinical samples

Blood samples of 300 μL from the subjects of two PK/PD studies of milrinone and dobutamine were collected from an indwelling arterial line into Na-heparin vials and centrifuged immediately. 50–100 μL plasma was stored at – 80 °C (up to 12 h storage at −20 °C prior to transfer to −80 °C was accepted) and transported on dry ice.

3. Results

3.1. Limit of quantification, linearity, carry-over, selectivity, accuracy and precision

The lower limit of quantification (LLOQ) was determined as 0.97 ng/ml for both analytes. A patient sample with near LLOQ concentration for dobutamine is shown in Fig. 2. At this level, interfering peaks for dobutamine can also be seen, however, the use of IS clearly identifies the analyte peak.

Fig. 2.

Chromatogram of a patient sample. Milrinone’s concentration is 113 ng/mL and dobutamine’s concentration is 4 ng/mL. IS concentration is 44 ng/mL. Interfering peaks on the dobutamine chromatogram were baseline separated from the dobutamine peak and did not interfere with quantitation.

The calibration set comprised 8 calibration points (prepared in duplicates), blank (containing IS) and double blank (blank plasma without IS). The back-calculated concentrations from the curve remained within ±15% of target values. Linearity, expressed as the coefficient of determination (R²), was >0.998 for both analytes. Carry-over did not present a problem, as both analytes and ISs were undetectable from blank injections after the highest calibrator. No interfering peaks for the analytes and the ISs were found in the double blank samples from six individual sources during the selectivity experiments. The data for accuracy and precision are presented in Table 3.

Table 3.

Accuracy and precision data (n = 5 for within-run accuracy; n = 15 for between-run accuracy).

Within-assay repeatability (Milrinone)
	LLOQ	QC3	QC2	QC1
Expected c (ng/mL)	1.10	3.20	76.51	223.88
N	5 of 5	5 of 5	5 of 5	5 of 5
Mean c (ng/mL)	1.08	3.11	80.86	231.73
Standard deviation	0.08	0.09	2.76	14.62
CV (%)	7.36	2.82	3.42	6.31
Accuracy (%)	98.07	97.11	105.69	103.51
Within-assay repeatability (Dobutamine)
	LLOQ	QC3	QC2	QC1
Expected c (ng/mL)	1.10	3.20	76.62	224.19
N	5 of 5	5 of 5	5 of 5	5 of 5
Mean c (ng/mL)	1.07	3.03	76.09	221.04
Standard deviation	0.04	0.12	3.48	14.39
CV (%)	4.08	4.05	4.57	6.51
Accuracy (%)	96.83	94.69	99.31	98.59
Between-assay repeatability (Milrinone)
	LLOQ	QC3	QC2	QC1
Expected c (ng/mL)	1.10	3.20	76.51	223.88
N	15 of 15	15 of 15	15 of 15	15 of 15
Mean c (ng/mL)	1.07	3.13	80.75	233.92
Standard deviation	0.07	0.09	2.01	20.65
CV (%)	6.55	3.0	2.5	8.83
Accuracy (%)	96.61	97.97	105.54	104.48
Between-assay repeatability (Dobutamine)
	LLOQ	QC3	QC2	QC1
Expected c (ng/mL)	1.10	3.20	76.62	224.19
N	15 of 15	15 of 15	15 of 15	15 of 15
Mean c (ng/mL)	1.05	3.02	76.61	229.44
Standard deviation	0.04	0.09	2.62	11.79
CV (%)	4.18	2.91	3.42	5.14
Accuracy (%)	95.20	94.22	99.99	102.34

3.2. Dilution integrity

Dilution integrity was evaluated for samples with concentrations higher than 300 ng/mL. Five replicates were tested at concentrations of 400 ng/mL and diluted 1:1 with blank plasma. For both analytes, the obtained accuracy for dilution integrity was within 96–105%.

3.3. Matrix effects

Matrix effects for both analytes were assessed using 6 different plasma sources at low and high concentration and described by matrix factor (MF). For dobutamine, MF ranged from 101 to 114%, and for milrinone between 90 and 103%. Results for hyperlipidemic plasma (with total cholesterol >240 mg/dL) for both analytes were between 101 and 106%. Additionally, haemolysed plasma did not significantly influence results as both analytes’ MF was between 89 and 98% and no clear trends with the increase of the level of haemolysis was observed for either analyte.

3.4. Freeze-and-thaw stability.

Freeze-and-thaw stability in low (LLOQ) and high (ULOQ) concentration varied from 100 to 110% for milrinone (Fig. 3), in both stabilised and unstabilised plasma, as well as for dobutamine in stabilised plasma. In unstabilised plasma, however, only 84% of dobutamine remained in low and 75% in high concentration samples (Fig. 3).

Fig. 3.

Bench top stability over 1 h, 2 h and 24 h, short term stability at fridge (4 ⁰C) for 24 h and freeze and thaw stability over three cycles for dobutamine and milrinone (error bars indicate the standard deviation of stability data).

3.5. Short term stability or bench-top stability.

In the case of milrinone, for both stabilised and unstabilised plasma, results were within 96–114%, without any observable trends (Fig. 3). For dobutamine (Fig. 3), in stabilised plasma, results ranged from 88 to 104%, with the exception of the high concentration sample, where only 68% of initial analyte concentration remained after 24 h. The results in unstabilised plasma show more severe degradation, as for both concentration levels only 19–84% of the initial concentration remained. Rapid degradation of dobutamine in unstabilised plasma was observed when samples were kept at room temperature for 24 h – with only 19% and 25% of initial concentration remaining at the lower and higher concentrations, respectively. (Fig. 3).

3.6. Long-term stability at −70 °C

For both analytes in stabilised plasma, long-term stability was within 97–113%. For milrinone, even in unstabilised plasma, this value was 111% at 3xLLOQ concentration level and 87% at ULOQ concentration level. For dobutamine, the long-term stability in unstabilised plasma ranged from 98% (at 3xLLOQ concentration level) to 109% (at ULOQ level).

3.7. 24 Hour stability in the autosampler at 4 °C

For both analytes, autosampler stability was 99–111% for stabilised and unstabilised plasma at all four (LLOQ, 3xLLOQ, MED and ULOQ) concentrations for both milrinone and dobutamine, demonstrating that the assay is suitable for reanalysis of the samples after 24 h and that samples are more stable after sample preparation. However, a significant decrease of 65% was observed in dobutamine and its internal standard peak areas during the autosampler stability study in unstabilised plasma samples. Additional stability experiments indicated a similar degradation rate when only either analyte or internal standard peak areas were compared. As low as 6 to 14% of the initial peak area was seen in the stability samples at 3xLLOQ and ULOQ levels, respectively, after storage on the autosampler. Yet, the accuracy of back-calculated concentration value of dobutamine was 102% at the 3xLLOQ level and 103% at ULOQ level when IS was considered.

3.8. Results from clinical studies

For milrinone, all 40 measured plasma concentration data points remained in the clinically relevant range and could, therefore, be used in population PK modelling. For dobutamine 119 study samples were analysed, dobutamine was not detected in 2 samples and remained below the LLOQ in 9 samples. Fig. 4 presents measured concentrations of milrinone and dobutamine in plasma samples from three patients. The results of PK studies will be published elsewhere.

Fig. 4.

Data from three patients dosed with milrinone (left) and dobutamine (right). For milrinone the dosing scheme included loading infusion of 0.73 μg/kg/min for 3 h (starting from time 0) followed by 0.16 μg/kg/min for 21 h. Dobutamine was administered escalating the dose from 5 μg/kg/min to 20 μg/kg/min, all concentration samples were taken 20–30 min after the start of the dose (steady state assumed).

4. Discussion

4.1. Method development and validation

A selective and sensitive method for simultaneous determination of milrinone and dobutamine, using the lowest known reported sample volume of 20 µL, was developed and validated for simultaneous measurement in neonatal plasma samples. The sensitivity of the method was achieved by using NH₄F as an eluent additive. The working mechanism and influence on sensitivity of NH₄F in negative ion mode for steroid-like methods is well known, but the same cannot be said for detection in positive ion mode. In LC-MS analysis, however, positive ion mode is generally preferred. During method development, comparison of eluent compositions revealed that use of NH₄F enhanced signal for both analytes in positive ion detection mode. For milrinone, the signal increase was two times that of the eluent, consisting only 0.1% formic acid, but for dobutamine, the enhancement was seven times greater. This signal enhancement mechanism is not yet fully described in the literature [25].

The method was fully validated and showed sufficient accuracy and precision for bioanalytical application, according to EMA guidelines [28], excellent linearity over the range of 1–300 ng/mL, no carry-over and excellent dilution integrity. Even when protein precipitation was chosen for the sample preparation, the assay did not suffer from matrix effects in the six different matrixes tested. Moreover, there was no significant influence on assay performance when haemolysed or hyperlipidaemic plasma was used for the analysis. Even though hyperlipidaemic plasma was not analysed as part of the current clinical study, the validated assay has been demonstrated to be suitable for different clinical scenarios.

4.2. Dobutamine stability

Analyte stability can be a major challenge in method development, as seen in this study. There is little information regarding dobutamine stability in the literature. Two publications [15], [16] acknowledge the existence of catechol-O-methyltransferase, which oxidizes dobutamine in blood. In previous studies, this problem was solved by performing sample preparation on ice [15] or by combining it with the addition of inhibitor glutathione [16]. However, no extensive stability research was performed in either case. In more recent research, there is no information on dobutamine degradation in human plasma samples. Thippani et al. did not observe any degradation in rat plasma [14]. Albóniga et al. likewise did not observe any degradation in pig K3-EDTA plasma, however, degradation in stocks was present [19].

Various options were explored to solve the stability issue. In dobutamine, the most likely cause are the two phenolic hydroxyl groups (Fig. 1), which undergo degradation. Phenols easily undergo autooxidation (sometimes even due to UV radiation), as well as oxidation due to enzymes. Therefore, 2 catechol-O-methyltransferase inhibitors – entacapone and tolcapone – were tested, although neither yielded sufficient results.

In further investigation, the antioxidants ascorbic acid and hydroquinone were tested. Ascorbic acid has previously proved to improve stability [30], and also provided the best results in this study. Samples prepared in plasma stabilised with ascorbic acid showed no significant change in IS reponse when injected at the beginning and end of a 100 sample run (Fig. 5).

Fig. 5.

Dobutamine IS response in stabilised and unstabilised plasma samples during analytical batch run.

When working with unstabilised plasma samples, even IS degradation was observed over the analysis time (Fig. 5). This suggests that whatever agent is responsible for the degradation of dobutamine, survives the sample preparation procedure in sufficient quantities to continue its work on dobutamine decay in processed samples and, consequently, affects autosampler stability. Since the degradation rate is the same for the dobutamine IS, accuracy and precision results for the stability experiments pass the acceptance criteria of ±15% when calculated via the IS ratio. While this certainly emphasises the importance of using the appropriate stable isotope-labelled IS, its protective effect commences only after it has been added to the sample. As the plasma inevitably must spend a certain amount of time without IS, for the sake of sensitivity, we recommend stabilising the plasma as soon as possible, i.e. before spiking or during thawing and to stabilise study samples during the sample collection.

4.3. Application to measurement of real samples

The method, utilising low sample volume and LLOQ, has great value for further PK and PD studies in both paediatric and neonatal populations. Even unstable analytes can be measured reliably by an experienced study team capable of following complex sample collection, storage and analysis procedures. Problems related to storage and handling of pre-added stabiliser-containing vials were overcome by adding the stabilising agent to the plasma during the thawing phase. Extensive stability testing was undertaken to ensure the validity of results.

While different modelling approaches are increasingly being used for dosing recommendations in paediatric populations [6], real-life data, at least in a limited number of patients, are necessary to test the validity of these approaches. In the milrinone study, plasma concentrations were consistent with previous studies applying similar dosing scheme [31]. For dobutamine, a standardised dosing regimen resulted in wide inter-patient variation in serum concentrations, which has been noted before in paediatric populations [32], [33]. As PD data of most cardiovascular drugs are extremely limited, further studies are needed to establish evidence-based therapeutic targets.

5. Conclusions

A highly sensitive simultaneous UHPLC-MS/MS method was developed for the simultaneous quantification of milrinone and dobutamine in small volume plasma samples. The use of NH₄F as an eluent increased the signal intensity twofold for milrinone and sevenfold for dobutamine, helping to achieve the required low limit of quantification of 0.97 ng/mL, using only 20 µL of blood plasma. Severe degradation of dobutamine was prevented by using ascorbic acid. Moreover, ascorbic acid should be added to the sample as soon as possible and, preferably, during sample collection.

The assay was used to analyse neonatal plasma samples under the clinical trials “The effect of milrinone on central and regional blood flow in preterm neonates undergoing patent ductus arteriosus ligation” (Eudra CT 2015-000486-31) and “Dose dependent effects of dobutamine on central and regional blood flow in preterm and term neonates” (Eudra CT 2015-004836-36).