A New Method for the Determination of Amisulpride in a Small Volume (200 μL) of Human Saliva Using LC-DAD Supported by SPE

Abstract

(1) Background: The concentration of amisulpride, an atypical antipsychotic drug, is most often determined in the blood, with many inconveniences. An alternative may be to use saliva as a diagnostic material for this purpose. The development of a method to determine amisulpride in saliva using a small volume of biological material could significantly improve patient comfort during Therapeutic Drug Monitoring (TDM). (2) Methods: Therefore, the aim of this study was to develop a method to determine amisulpride in 200 μL of saliva using solid-phase extraction for isolation and liquid chromatography with a diode array detector (LC-DAD) for quantitative analysis. (3) Results: The method was validated by determining its linearity in the concentration range 5–500 ng/mL (R² > 0.99), and the intra- and inter-day precision expressed as coefficient of variation (CV%) did not exceed 9%. (4) Conclusions: The developed method was used to determine the salivary concentration of amisulpride in patients treated with the studied compound, confirming its usefulness in TDM.

1. Introduction

Amisulpride is an atypical antipsychotic drug belonging to the benzamide derivatives (Figure 1). The drug has a selective affinity for dopamine receptors, with a high affinity for D2 and D3 receptors. As an antagonist of dopamine receptors, it blocks them in the postsynaptic part, producing an antipsychotic effect. By acting on the presynaptic dopamine D2 and D3 receptors, amisulpride shows a relieving effect on the negative symptoms that occur. The potency of the drug depends on the dose administered [1].

The bioavailability of amisulpride after oral administration is 48%, and the half-life is 12 h. A high-fat diet does not affect the absorption of the substance, whereas meals containing high amounts of carbohydrates have a significant effect on pharmacokinetic parameters, causing delayed or reduced absorption of the drug. After absorption, amisulpride binds only slightly to plasma proteins (16%). No interaction with other drugs at the level of distribution has been observed. In the body, the drug is metabolised slightly to two metabolites that do not show pharmacological activity [2].

Amisulpride is primarily used in the treatment of schizophrenia and is given when positive symptoms are predominant [2]. The drug has also been used to prevent nausea and vomiting in the postoperative period [3,4].

Although amisulpride belongs to the group of atypical neuroleptics, quite often extrapyramidal symptoms appear after its administration [2], which in patients with Parkinson’s disease may lead to an exacerbation of the disease symptoms. In addition, other side effects are likely to occur, the most serious of which is a malignant neuroleptic syndrome, characterised by, among others, hyperthermia, impaired consciousness, increased muscle rigidity and autonomic nervous system dysfunction. The use of amisulpride affects QT (the electrical properties of the heart calculated as the time from the start of the Q wave to the end of the T wave) prolongation, which is associated with an increased risk of severe ventricular arrhythmias. In patients over 65 years of age, the drug should be administered with extreme caution due to the greater likelihood of blood pressure falls or excessive sedation. In addition, a lower dose than usual may be necessary for renal insufficiency [2].

The primary biological material in which amisulpride is labelled is blood, either whole blood [5,6,7], plasma [6,8,9,10,11,12,13,14,15,16,17,18,19] or serum [5,9,13,16,20]. Few publications report the use of other biological matrices, such as saliva [5,6,16,19,21] or breast milk [17]. Quantitative analysis of amisulpride was carried out using liquid chromatography, which was usually preceded by isolation by liquid-liquid extraction (LLE) [5,6,8,11,12,13,14,15,16,19,21]. In some cases, solid-phase extraction (SPE) [7,10,15,20], deproteinisation [17,18] or column switching [9] were also used.

Liquid chromatography is the most commonly used method to determine the concentration of amisulpride in biological material. For the detection of the analyte, it is coupled to an MS/MS [5,6,7,11,12,13,14,16,17,18,21], which is a very sensitive detector. However, at the same time, the main limitations of using MS/MS detection include high costs, the requirement of having qualified and experienced analysts and the need for very high-purity solvents. This limits the use of this detector in routine studies. Therefore, a cheaper solution is to use a UV detector [8,9,20], which is widely available, and the structure of amisulpride allows the detection of the compound with a spectrophotometric detector even when the concentration of the compound is at 10 ng/mL [9].

The use of blood for amisulpride analysis offers the possibility of determining the total concentration of the drug in the body, as well as controlling its pharmacokinetics. However, blood collection is associated with discomfort for the patient as well as possible infectious complications. The collection itself requires appropriate, sterile equipment and the presence of qualified personnel. For other biological materials, such as saliva, whose collection is easier and non-invasive, a concentration correlation between blood and saliva is desirable. In the case of amisulpride, a study by Fisher indicates that saliva may be a potential diagnostic material for therapeutic drug monitoring (TDM) [6]. When determining amisulpride in saliva, attention should also be paid to the volume of samples collected and the method of collection [19,21]. In the case of dry mouth, a sample of 0.5 mL or more may be difficult to obtain. Therefore, when developing a method for the determination of amisulpride in saliva, it would be necessary to develop one that uses as small a sample volume as possible. To date, isolation of amisulpride from saliva has been carried out using LLE, which, although allowing efficient sample purification, requires volatile and toxic solvents [5,6,16,19,21]. In addition, inappropriate solvents can cause emulsions to form during the extraction process. It is now possible to use supported liquid-liquid extraction (SLE), which is performed in columns. This reduces the formation of emulsions during the process. However, lipophilic and often toxic solvents are also used in this method. These solvents, used even in a small volume but over a long period when testing multiple samples, can cause discomfort to the investigator due to their penetration into the central nervous system.

Therefore, the aim of this study was to develop an efficient method for the determination of amisulpride in a small volume (200 μL) of saliva sample using SPE and liquid chromatography coupled to a DAD detector and then to apply the developed method to determine the concentration of the analyte in the saliva of patients treated with amisulpride.

2. Materials and Methods

2.1. Chemicals and Solvents

Stock solution (1 mg/mL) of amisulpride was purchased from Sigma-Aldrich (St. Louis, MO, USA). The internal standard (IS) chlordiazepoxide was purchased from W Polfa Tarchomin (Warsaw, Poland). Methanol and acetonitrile of HPLC gradient grade were obtained from POCh (Gliwice, Poland). Reagent-grade formic acid (98–100%), ammonia solution 32% (v/v) (NH₄OH), and potassium hydrogen phosphate (K₂HPO₄) were purchased from POCh (Gliwice, Poland). Water was purified by Ultra-Toc/UV system, Hydrolab (Straszyn, Poland). For solid-phase extraction columns, Isolute with C18 cartridges (1 mL; 25 mg) were purchased from Biotage (Hengoed, UK).

2.2. LC-DAD Conditions

Chromatographic analysis was performed using a Nexera XR UHPLC liquid chromatograph (Shimadzu, Kyoto, Japan) comprising a CBM-20Alite control system, LC-30AD pump, CTO-20AC thermostat, SIL-30AC autosampler, SPD-M30A UV-VIS detector with diode array and SPD-M30A high-sensitivity measuring cell (85 mm). Chromatographic separation was performed using Nucleosil 100-5 (C18; 125 mm × 4 mm, 5 µm; Knauer, Berlin, Germany) with precolumn, maintained at 35 °C. Acetonitrile and 0.1% formic acid were used as the mobile phase components, at a flow rate of 1 mL/min, with the following gradient: starting with 10% of acetonitrile and finishing with 65% of acetonitrile. The total run time of analysis was 12 min.

2.3. Preparation of Quality Control (QC) and Standard Samples

A standard solution of chlordiazepoxide (internal standard, IS) (10 mg/mL) was prepared by dissolving 10 mg of the substances in methanol. The stock solutions were used for the preparation of calibrators and QC samples. Working solutions of analytes were prepared by diluting stock solutions of amisulpride and IS in methanol. The working solution of IS (100 μg/mL) was prepared by diluting 10 μL of the stock solution in 990 μL of methanol. Then, 100 μL of 100 μg/mL IS solution was mixed with 900 μL of methanol. The calibration standard of amisulpride (100 μg/mL) was prepared by mixing 100 μL of 1 mg/mL amisulpride solution with 900 μL of methanol. Additional dilutions, at concentrations of 10 and 1.0 μg/mL, were prepared by mixing 900 μL of methanol and 100 μL of 100 or 10 μg/mL solutions, respectively. The standard stock and working solutions were stored at −21 °C.

Calibration samples were prepared as follows: blank saliva samples were spiked with 50 μL of IS (10 μg/mL) and amisulpride working solutions (0.1, 1 and 10 μg/mL) for amisulpride concentrations of 5, 10, 50, 100, 150, 300, 500, 750, 1000, 3000 and 5000 ng/mL, as appropriate.

For QC solutions, 200 μg/mL working solutions were prepared by diluting 20 μL of amisulpride stock solution in 980 μL of methanol. Working solutions at concentrations of 20, 2 and 0.2 μg/mL were prepared by mixing 900 μL of methanol and 100 μL of 200, 20 or 2 μg/mL working solution, respectively. QC samples were prepared as follows: blank saliva samples were spiked with 50 μL of IS (10 μg/mL) and amisulpride working solutions (0.2, 2, and 20 μg/mL) for amisulpride concentrations of 25, 400, 2500, and 4000 ng/mL.

2.4. Collection of Saliva Samples

Saliva samples were collected with Salivettes^® (Sarstedt, Nümbrecht, Germany). Before the process, volunteers were required to refrain from eating for at least half an hour and 10 min before sampling to rinse their mouths with water. Salivettes were placed in the mouth for 2 min and then centrifuged at 8000 rpm for 5 min. The obtained saliva was frozen and stored at −21 °C until analysis.

2.5. Extraction of Amisulpride from Saliva Samples

For analysis, 200 μL of blank saliva sample was collected and transferred into plastic tubes, diluted with 0.5 mL of 0.1 M K₂HPO₄ solution, and mixed. Next, appropriate amounts of amisulpride and IS standards were added, and the samples were again vortex-mixed. Then the samples were loaded onto SPE columns, which had been pre-conditioned with 0.5 mL of methanol and 0.5 mL of water. The cartridges were washed with 0.5 mL of the mixture of methanol and water (10:90, v/v) and dried for 10 min. Analytes were then eluted with 0.5 mL methanol. The eluent was dried at 37 °C under a nitrogen stream, and the residue was reconstituted in 100 μL of a mixture of 0.1% formic acid and acetonitrile (90:10, v/v), vortex-mixed, and transferred to autosampler vials. Finally, 20 μL of the sample was injected into the chromatographic column.

2.6. Validation

The validation process was performed in accordance with the EMA guidelines [22].

2.6.1. Linearity

The linearity was verified by preparing four calibration curves (range 5–5000 ng/mL) on four different days. Acceptance criteria were coefficient of determination (R²) ≥ 0.99 and residuals ≤15% at each concentration. Only at the lower limit of quantification (LLOQ) were the residuals ≤20% accepted.

2.6.2. Intra and Inter-Day Precision

Method precisions were evaluated using four analyte concentrations (25, 400, 2500 and 4000 ng/mL). For intra-day precision, each concentration was analysed in five replicates within one day. The inter-day precision was determined by performing five analyses of each concentration for four consecutive days (n = 20). The precision of the method was expressed in terms of imprecision by calculating the CV% of pooled within-day and between-day. Imprecision was deemed acceptable for CV ≤ 15%.

2.6.3. Limit of Detection (LOD) and Lower Limit of Quantification (LLOQ)

LOD was defined as the lowest concentration of analyte that could be identified, with a signal-to-noise ≥ 3. LOD was empirically determined by spiking saliva samples with decreasing analyte concentrations. Each concentration studied was analysed in triplicate.

LLOQ was defined as the lowest concentration that could be quantified with a signal-to-noise ≥10, adequate precision (coefficient of variation (CV%) <20%), and accuracy (target concentration ± 20%). LLOQ was evaluated by analysing five replicates.

2.6.4. Selectivity

The selectivity of the method was determined by excluding the presence of endogenous interferents. For this purpose, blank saliva samples from 10 different donors were studied and subjected to the extraction procedure described in Section 2.5 and analysed under the chromatographic conditions presented in Section 2.2. The absence of interferents allowed us to conclude that the method was selective.

2.6.5. Extraction and Absolute Recovery

Method recovery was determined for four concentration levels (25, 400, 2500 and 4000 ng/mL) by analysing each concentration six times. The absolute recovery was performed by comparing the peak areas of the extracted analyte with the peak areas of the six neat standards. The extraction recovery was determined by comparing the areas of the extracted analyte to those obtained from ten blank post-spiked samples. In both cases, an acceptable result was considered to be a value exceeding 50% of the average value of six neat standards or the post-spiked samples at each concentration.

2.6.6. Stability

Amisulpride stability was tested for four concentrations (25, 400, 2500, and 4000 ng/mL). The stability at each concentration was determined both in the matrix during storage at 8 °C and in a freeze–thaw test when spiked saliva samples were stored at −21 °C. In both cases, for each concentration of amisulpride, three 800 μL saliva samples were prepared in plastic tubes, spiked with an appropriate volume of an analyte, and mixed. Then, 200 μL of saliva was taken from each sample, and IS was added, extracted, and chromatographed according to the procedures described in Section 2.5 and Section 2.2. The remaining volume of the sample was placed back in the refrigerator or frozen and analysed in the following days. If the concentration of amisulpride decreased by less than 15% under the given storage conditions, it was considered to be stable.

2.7. Clinical Application

The developed method was applied to determine the concentration of amisulpride in saliva obtained from patients treated with amisulpride (200 to 400 mg/d). Saliva samples were obtained from 6 women in the morning, 3 h after the amisulpride administration. The samples were collected from patients of the Nervous and Mentally Ill Hospital in Starogard Gdanski (Poland). The study protocol was approved by the ethical committee of the Medical University of Gdansk, Poland (NKBBN/393/2021; 25 June 2021).

3. Results

3.1. Chromatographic Analysis

To optimise the chromatographic separation process, the mobile phase composition and its flow rate were selected. This ensured adequate separation of the analyte and internal standard from the other matrix components. To improve the chromatographic analysis of saliva, it was decided to use a gradient to elute lipophilic molecules from the matrix that strongly interact with the stationary phase. The chromatographic analysis time was 12 min, and peak detection was carried out at 280 nm. Chlordiazepoxide was used as an IS, which exhibits similar physicochemical properties, resulting in similar retention times and adequate UV absorption at 280 nm. A chromatogram of the standard solutions using the optimised conditions is shown in Figure 2.

3.2. Solid Phase Extraction

Optimisation of the extraction process took place in several stages. At first, a procedure was developed to purify blank saliva samples to maximise the removal of ballast from the biological matrix. Hence, saliva samples were diluted with a mixture of methanol and water (1:1; v/v) or 0.1 M K₂HPO₄ solution, shaken, and loaded onto activated columns. The cartridge was then washed using redistilled water, 2% formic acid, or a mixture of methanol and water (10:90, v/v). In addition, two-stage washing was also used, where the sorbent was washed with water or 2% formic acid in the first stage and with a mixture of water and methanol (1:1; v/v) in the next stage. Subsequently, the sorbent was dried, and the residue was eluted using methanol, acetonitrile or their alkaline modification in the form of a 5% ammonia solution. The procedures described are presented in

Table 1. Procedures used to examine the efficacy of solid phase extraction (SPE).

Procedure	Dilution Solvent	Washing Solvent	Elution Solvent
1.	methanol:water (1:1)	water	methanol
2.			acetonitrile
3.		water methanol:water (1:1)	methanol
4.			acetonitrile
5.		2% formic acid	5% ammonium in methanol
6.			5% ammonium in acetonitrile
7.		2% formic acid methanol:water (1:1)	5% ammonium in methanol
8.			5% ammonium in acetonitrile
9.	0.1 M K2HPO4	methanol:water (10:90, v/v)	methanol

.

All procedures were then applied to isolate amisulpride from spiked saliva samples. For this purpose, samples were prepared at concentrations of 25, 400, 2500 and 4000 ng/mL. Each concentration was analysed in triplicate. Procedures in which a mixture of methanol and water (1:1) was used to dilute the sample, where the analytes were eluted from the columns with acetonitrile or its mixture with ammonia, did not ensure adequate sample purification. The consequence was peaks of matrix-derived impurities during the retention time of the analytes in the sample blank extracts.

In procedures where a methanol/water mixture (1:1) was used to dilute the samples and methanol was used to elute the analytes, the blank sample extracts were better purified. Therefore, it was decided to check the linearity of procedure 3. However, the results obtained were not reproducible in terms of extraction efficiency. For this reason, it was decided to analyse chromatographically the eluates collected at each extraction step. These showed losses of analyte at the sample application and sorbent washing stages. Taking into account these observations and the physicochemical properties of amisulpride, the procedures developed so far were analysed, and the extraction procedure used (procedure 3) was modified by selecting appropriate solvents.

The next step in the work was the extraction of amisulpride to the solid phase after diluting the sample with 0.1 M K₂HPO₄ solution (procedure 9), washing the sorbent with a mixture of methanol and water (1:9) and eluting the analytes with methanol. Figure 3 shows the chromatogram of the blank saliva sample and the extract of the saliva sample loaded with 50 and 300 ng/mL of amisulpride.

3.3. Method Validation

The linearity of the method was examined in the range of 5–5000 ng/mL. The results of the study confirmed the linearity of the method, and it meets the designated acceptance criterion (R² ≥ 0.99,

Table 2. Calibration curve parameters for the developed method (linearity in the range 5–5000 ng/mL).

Calibration Curve y = ax + b (n = 4)
Range (ng/mL)	5–5000
Determination coefficient (R2)	0.9978 ± 0.0021
Slope a ± Δa	0.0005 ± 0.00005
Intercept b ± Δb	−0.0147 ± 0.00700
LOD (ng/mL)	3.0
LLOQ (ng/mL)	5.0

). The LLOQ for amisulpride was set at 5 ng/mL. The determined validation parameters, the results of which are presented in

Table 3. Validation parameters for the developed method. Stability of amisulpride at four QC concentrations after storage at 8 °C and −21 °C, expressed as % loss.

QC (ng/mL)	Intra-Day (CV%)	Inter-Day (CV%)	Extraction Recovery (%)	Absolute Recovery (%)	Stability (Difference %)
					8 °C	−21 °C
25	4.79	8.84	101.22	61.03	−2.50	−0.50
400	4.41	4.72	91.81	83.10	−2.81	−1.24
2500	5.05	7.44	92.82	80.69	−2.63	−1.65
4000	1.79	4.95	96.02	79.91	−3.89	−2.49

, indicate that the developed method is precise, with CV values indicative of this being between 1.79 and 8.84% for both inter- and intra-day precision.

The selectivity of the method was also determined by analysing 10 blank saliva samples. It was found that there were no peaks of matrix-derived substances with retention times close to that of the analyte and IS. On this basis, the method was found to be selective.

3.3.1. Extraction and Absolute Recovery

Both extraction recovery and absolute recovery were investigated for four concentrations of QC (25, 400, 2500 and 4000 ng/mL), and the results obtained are shown in Table 3. In the case of extraction recovery, the areas of the analytes extracted by the developed method were compared with the peak areas of blank saliva samples spiked after the extraction process, and the recovery results obtained ranged between 91.81 and 101.22%. To determine absolute recovery, the peak areas of the analytes after extraction were compared with the peak areas obtained during the analysis of six neat standards at each concentration. The lowest absolute recovery value of 61.03% was found at a concentration of 25 ng/mL.

From the results obtained, it can be concluded that both extraction recovery and absolute recovery meet the acceptance criterion (>50%) for all concentrations studied.

3.3.2. Stability

The stability of amisulpride was tested for four concentrations of solutions after extraction, which were stored at 8 °C and −21 °C. The results of the study are presented in Table 3. Data on the stability are presented as a loss of concentration relative to the concentration determined on day one and were expressed as % loss. The results presented indicate that amisulpride is stable under the conditions investigated.

3.4. Clinical Application

Saliva samples were from six patients treated with amisulpride at a dose of 200 or 400 mg per day. In all samples, the developed method allowed the determination of amisulpride. An example of a chromatogram of a patient’s saliva extract is shown in Figure 4, while the determined concentration and details of the patients are included in

Table 4. Patient’s data. Concentrations of amisulpride found in saliva of six female patients.

Patient	Age	Dose (mg/Day)	Amisulpride (ng/mL)
1	58	400	2852.9
2	62	400	4713.1
3	56	400	4707.6
4	40	200	216.3
5	38	200	543.3
6	43	200	104.8

.

4. Discussion

Amisulpride, although it belongs to the atypical neuroleptics, which have a higher safety profile than older generation neuroleptics, can cause adverse effects. They usually appear earlier than the therapeutic effect, which may further influence patient discontinuation of therapy. In addition, the incidence and severity of side effects were found to be correlated with the drug’s blood concentration. Studies also indicate that the blood concentration of the drug will be affected by factors such as the gender and age of patients [23,24]. Therefore, control of amisulpride concentrations in the body is indicated, in particular, in women, in whom concentrations are higher, because of differences in weight, renal function, and volume of distribution [25]. TDM is also indicated if patients are taking other preparations, e.g., lithium or clozapine, or if they smoke [24].

The aim of the study was to develop a method to determine amisulpride in a small volume of saliva using solid-phase extraction and liquid chromatography. In the studies published to date, isolation of amisulpride from saliva was performed using LLE. In contrast, SPE was only used to extract this compound from blood. The method developed allowed the determination of amisulpride in saliva from a very small volume of biological material (200 μL). The use of such a small volume of saliva allows the determination of amisulpride even when the patient suffers from a dry mouth.

A comparison of the results of the methods developed to date for the determination of amisulpride in a biological matrix is shown in

Table 5. A summary of recent data on amisulpride analysis in human saliva and other biological material.

Biological Matrices	Volume of the Sample (µL)	Isolation	Separation and Detection	LOQ (ng/mL)	Linearity (ng/mL)	R2	Concentration (ng/mL)	References
Plasma	1000	LLE	HPLC-UV	20	100–1000	0.985	107–14,824	[8]
	1000	SPE	HPLC-FL	2	2–800	>0.99	$\overline{x}=$ 424.4 ± 292.8	[10]
	50	LLE	LC-MS/MS	0.5	0.5–500.52	=0.9999	522.58	[11]
	100	LLE	LC-MS/MS	0.1	0.1–100	>0.99	-	[12]
	100	LLE	HPLC-MS/MS	2	2–2500	>0.99	$\overline{x}=$ 947.9 $\overline{x}=$ 1093.4	[14]
	1500	SPE	LC-FL	10	10–1000	=0.9979	$\overline{x}=$ 501 ± 216 $\overline{x}=$ 515 ± 208	[15]
	20	deproteinisation	UPLC-MS/MS	20	20–2000	>0.9977	21.9–773.7	[18]
Plasma/breast milk	plasma 25 milk 15	deproteinisation	LC-MS/MS	breast milk 0.6 plasma 0.5	breast milk 0.6 to 75 plasma 0.5–360	>0.999	maternal plasma 95 breast milk 1131 baby’s plasma 10	[17]
Serum, plasma	200	LLE	LC-MS/MS	0.5	1–400	>0.98	-	[13]
	100	column-switching	HPLC-UV	10	10–600	>0.99	-	[9]
Serum	1000	SPE	HPLC-UV	10	10–1000	>0.9995	-	[20]
Blood	500	SPE	HPLC-MS/MS	5	5–500	>0.99	-	[7]
Whole blood, oral fluid, serum	200	LLE	LC-MS/MS	10	10–500	>0.99	-	[5]
Plasma, oral fluid, whole blood	500	LLE	HPLC-MS/MS	-	-	-	plasma 193,241; oral fluid 190, 208; whole blood 243, 381	[6]
Plasma, serum, oral fluid	200	LLE	LC-MS/MS	6	10–500	>0.99	plasma/serum 413–2348	[16]
Plasma, oral fluid	500	LLE	LC-MS/MS	5	5–800	>0.99	plasma 40–1338 oral fluid 16–1827	[19]
Oral fluid	500	LLE	UPLC-MS/MS	3.20	3.20–1920	0.995	1.04–68.66	[21]

. In studies published to date, 500 μL of saliva has usually been collected for analysis [6,19,21]. Although Fisher et al. collected 200 μL of biological material for determination, the utility of this method has not been confirmed for the determination of amisulpride in patients’ saliva. The use of 200 μL of saliva also allows the determination of amisulpride if the patient suffers from a dry mouth or the collection of a larger volume of saliva is troublesome, e.g., if the patient is disturbed and has difficulty spitting a larger volume of saliva into a vessel.

The developed SPE-LC-DAD method has good linearity over a wide concentration range (5–5000 ng/mL; R² > 0.99). The selection of the concentration range was based on the sparse literature data on amisulpride concentrations in patients’ saliva and also taking into account the physicochemical properties of the compound, primarily pKa [21]. The selected concentration range coincides with levels determined in saliva samples from patients (104.8–4713.1 ng/mL). The developed method has a high precision of determination, as evidenced by low CV values below 10%. Smaller concentration ranges for linearity determination were most often chosen when the method developed for the determination of amisulpride in saliva was not used to analyse patient samples. The few publications that concern the determination of amisulpride in saliva suggest that there is a correlation between drug concentration in saliva and plasma [6]. Therefore, the selected linearity range of the method studied also coincided with the range of concentrations found in plasma.

The use of SPE for sample purification allowed the efficient isolation of the analyte and IS from the biological matrix. To our knowledge, SPE has previously been used to isolate amisulpride from blood but has not been used for extraction of this compound in saliva. The extraction we used provided a high analyte recovery of >91%. In addition, we avoided the use of large volumes of volatile and toxic solvents that are used in liquid-liquid extraction. Literature data indicate that for the LLE used to isolate amisulpride from saliva, the recovery was approximately 40% [16,21]. In contrast, for SPE used to isolate amisulpride from blood, the recovery exceeded 90% [15,20]. It can therefore be concluded that the SPE procedure developed and used is efficient and can be applied to the analysis of amisulpride in saliva.

Previous literature reports indicate that amisulpride is stable in saliva both during storage at 8 °C and after freezing [5]. Our study also confirmed its stability in saliva under all conditions tested, i.e., 8 °C and −21 °C.

The method developed was used to determine amisulpride in the saliva of patients treated with the studied compound at a dose of 200 or 400 mg/d. The few literature data on the determination of amisulpride in saliva are consistent with the range of concentrations determined in our study [6,21]. Amisulpride is a weak base and is characterised by a high pKa coefficient (9.4), readily diffuses into saliva [21] and reaches high concentrations in saliva. Its correlation coefficient between plasma and oral fluid is approximately 1:1 [6], suggesting that saliva may provide an alternative to blood when monitoring a patient’s drug intake when a non-invasive collection of a small volume of samples is needed.

5. Conclusions

In the study, a simple and fast method for the isolation of amisulpride from saliva by solid-phase extraction was developed and optimised. Among the procedures tested, the one in which the sample was diluted with K₂HPO₄ solution and, after loading onto the columns, the sorbent was washed with the mixture of methanol and water and eluted with methanol, proved to be the most favourable. The developed method requires a very small sample volume (200 µL), and the developed procedure was applied before LC-DAD quantitative analysis. The present SPE-LC-DAD method was linear in the range of 5–5000 ng/mL (R² > 0.99). It also has high precision, as evidenced by the fact that the highest CV value was 8.84%. Amisulpride was stable under the conditions tested. The developed SPE-LC-DAD method was also used to determine the concentration of the drug in the saliva of the patients. Amisulpride concentrations were determined in all samples tested. Therefore, it can be assumed that the developed method can be used to monitor the concentration of amisulpride in the saliva of patients.