2.1 Materials

Sigma-Aldrich Chemical Company (St. Louis, MO, USA) supplied the l-menthol, ethylene glycol (EG), salicylic acid (SA), (1S)-(+)-camphor-10-sulfonic acid (CSA), (BA), methyltrioctylammonium chloride (MTOAC), n-butanol, 1-undecanol, n-butanoic acid, codeine phosphate and morphine sulfate. 1-decyl-3-methylimidazolium chloride ([DMIM]Cl) and sodium dodecyl sulfate (SDS) were supplied by Merck (Darmstadt, Germany). A 1000 mg/L stock solution of morphine and codeine was prepared with a water: methanol (1:1 v/v) ratio. The necessary test solution was prepared from stock daily with adequate dilution.

2.2 Instrumentation

Morphine and codeine were examined by a Knauer high-performance liquid chromatography system (Germany) attached with an H5-ODS C18 column, binary pumps, a variable wavelength UV detector and Chromgate software (version 3.1). A Rheodyne model 7725i manual sample injector with a 20 μL loop was used for injection. The isocratic elution technique used a mobile phase of 10.0 mM Na₂HPO₄ and 0.70 mM SDS (pH = 6.5). The flow rate used was 1.0 mL per minute. The analytes has absorbance at 285 nm, hence used for the detection.

2.3 Sample collection and preparation

With informed consent, blood samples for a blank study were taken from a healthy 28-year-old male volunteer who had not used medication or opium in at least six months. The clinical microbiology lab at local Hospitals, supplied a real plasma sample from a 43-year-old man who had surgery and was administered morphine and codeine. The samples were preserved at −18 °C before analysis.

The sample was prepared by incubating 1.0 mL of plasma as received from the hospital without dilution was mixed with 800 μL of 15 % w/v ZnSO₄ in a test tube at 4 °C for 10 min. The acquired samples were centrifuged at 4000 rpm to prevent any matrix impact. The obtained supernatant was carefully transferred to a new, clean container and diluted with 10 mL of distilled water. The analyte in these solutions was investigated in this study.

2.4 Preparation of deep eutectic solvents

Table 1 shows the seven DESs synthesized based on previous research, their switchability, and references. DES-1 − l-menthol: ethylene glycol (1:1 M ratio); DES-2 − l-menthol: salicylic acid (4:1 M ratio); DES-3 − l-menthol: camphor sulphonic acid (5:1 M ratio); DES-4 − l-menthol: phenol (1:1 M ratio); DES-5 − MTOAC:n-butanol (1:1 M ratio); DES-6 − [DMIM]Cl:1-undecanol (1:2 M ratio) and DES-7 − [DMIM]Cl:n-butanoic acid (1:2 M ratio) (Abri et al., 2019; Ahmadi Jouybari et al., 2023; Raj, 2020; Pirsaheb and Fattahi, 2018).

2.5 Extraction procedure

For extraction, about 10.0 mL of the dilute plasma sample (which may or may not spike with morphine and codeine) was mixed with 50 μL of selected DES-3 in a 20 mL test tube. Addition of 100 μL of KOH solution (5 mol/L) and mixed for 3 min by shaking produced a single-phase DES and sample system. Following the drop-by-drop addition of 95 μL of HCl (5 mol/L), the target analytes and DES-3 were extracted as a single phase on the sample solution surface without centrifugation. The extracted phase was solidified by immersing the sample tube in an ice-cold bath for 5 min. The solidified phase was collected in a clean container, allowed to liquefy at room temperature, and injected into the HPLC system for analysis. The pH-switchable DES-LPME approach for quantitative analysis was assessed with the following parameters: accuracy, precision, repeatability along with intra- and inter-day repeatability, LR (linear range), LOD (limit of detection), LOQ (limit of quantification), ER (extraction recovery), and EF (enrichment factor).

3.1 Deep eutectic solvent selection

DESs interact differently with different analytes because of variations in polarity, structure, and other physical and chemical properties. Herein, seven different DESs were developed and tested for extracting morphine and codeine from diluted plasma samples using pH-switchability (Fig. 1). Table 1 illustrates that three DESs (DES-5, DES-6, and DES-7) were eliminated from the study due to their inability to turn on or off pH. The other DESs are effective at the molar ratio of l-menthol: CSA is 5:1. It is noted from Table 1 that DES-3 was found to be a suitable extractant based on its efficacy.

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Fig. 1

The effect of the DES type (A) and volume of DES (B) on the extraction recovery of morphine and codeine in blood samples obtained from DES–LPME/HPLC–UV.

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3.2 Deep eutectic solvent volume selection

One important factor in liquid-phase microextraction that needs to be carefully studied is the extraction solvent volume. However, it should not be set so high that it reduces the enrichment factor. The influence of DES volume on morphine and codeine extraction recovery was tested by varying the volume from 30 to 150 μL. Fig. 2(B) shows that raising the volume of DES from 30 to 50 μL enhanced the ER of the required analytes. Additional DES volume increases will keep the extraction recovery steady or slightly lower. The decrease in extraction efficiency is likely related to variations in the alkali utilized (100 μL) and problematic extractant phase collection with increasing DES volume. Thus, 50 μL DES was selected as the optimal volume.

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Fig. 2

The effect of the KOH concentration (A) and volume of HCl (B) on the extraction recovery of morphine and codeine in blood samples obtained from DES–LPME/HPLC–UV.

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3.3 KOH concentration selection

DESs used in this study are hydrophobic and immiscible with water. The present process transfers the DES into the sample system without needing a vortex, ultrasonic, or disperser solvent. As a result, the analytes' surface area in contact with the extraction solvent increases significantly, increasing extraction recovery. High concentrations of KOH are inefficient for achieving phase separation, while low concentrations do not show phase transition. Hence, KOH concentrations were optimized. Experiments were conducted with 100 μL of KOH at 1 to 10 mol/L concentrations. Fig. 2(A) shows that KOH concentrations below 3 mol/L do not result in phase transition or solvent transfer into the aqueous phase. The system is converted into a single phase during extraction by raising the KOH concentration from 3 to 5 mol/L. When the concentration is between 5 and 7 mol/L, the recommended volume of HCl is insufficient for phase separation, resulting in lower ER. Thus, the optimal KOH concentration was chosen as 5 mol/L.

3.4 HCl volume selection

From section 3.3, KOH concentration (7 mol/L) was preferred for the phase transition and single-phase system. Different HCl solutions (7 mol/L) with different volumes were used to neutralize the environment's alkalinity and separate phases. The phase separation was achieved by gradually increasing the HCl volume from 75 to 95 µL. Fig. 2(B) shows that the volume less than 75 µL does not result in phase separation. Strikingly, the phase separation gradually occurs at 95 µL, but the extraction recovery reduces with increasing HCl volume. Thus, 95 µL of the HCl was selected for testing.

3.5 Influence of salt

Analytes become less soluble in water and more inclined when salt is added to an aqueous solution. These two mechanisms partially neutralize the effects of salt on liquid-phase microextraction efficiency. The proposed procedure involves mixing acid-base and adjusting pH to obtain a high-concentration KOH solution. It is reasonable to assume that increasing the salt concentration does not influence extraction efficiency. However, a series of studies were conducted using NaCl solution at 0 to 5 % (w/v) concentrations. The findings of the studies that were carried out without the addition of NaCl showed that the addition of varied amounts of NaCl did not affect the analyte extraction recovery. Therefore, the subsequent studies were carried out in the absence of NaCl.

3.6 Influence of extraction time

According to the method outlined, the ER is considered as the interval between adding the KOH solution and starting to add the HCl solution. To examine the impact of extraction time (ET) on the recovery of morphine and codeine, we utilized an ET of 0 for the sample solution, resulting in a single-phase system. The extractant takes over 30 s for complete miscibility with the aqueous solution, and hampers ER due to the unavailability of the high contact surface. Intervals longer than 30 s have minimal influence on ER. Thus, the ideal extraction time was set at 30 s.

3.7 Quantitative analysis

An overview of the parameters used to construct the calibration curve is given in Table 2. The linear range of morphine and codeine was achieved by spiking blank blood with varying dosages of target analytes, and the samples were examined in triplicate. While considering days as a coefficient, the inter-day studies revealed a linear range of 1.5–300 μg L^-1. It was accomplished by adding predetermined doses of codeine and morphine to the defined concentration of blood samples. Following analyte extraction and HPLC analysis with seven replications, the analytes were determined to be 3.7–4.3 % and 5.4–6.2 %, respectively. An accuracy of 93.0 − 107.0 and 91.8 − 106.0 % was found for the inter-day and intra-day, respectively. The signal-to-noise ratio of 3 (S/N = 3) resulted in LODs of 0.5 µg/L, whereas S/N = 10 gave LOQs of 1.5 µg/L. At 100 μg L^-1, the EF and ER of morphine and codeine were found to be 152–166 % and 76–83 %, respectively.

3.8 Real samples analysis

After optimizing all parameters and creating a calibration plot, the method was tested on real blood samples. Initially, a blank blood sample free from drugs was collected from a 28-year-old healthy male volunteer and evaluated using the pH-switchable DES-LPME method. According to the results, the analytes were absent in the blank sample collected from the healthy male volunteer. Next, a blood sample from a 61-year-old opium user was collected and evaluated using the current approach. The samples tested positive for morphine and codeine at concentrations of 158.5 and 78.2 μg.L^-1, respectively. Furthermore, blood samples from two patients − a 25-year-old male patient and a 43-year-old female patient − who were undergoing morphine and codeine treatment showed the presence of both analytes. The results are presented in Table 3.

Real blood samples were tested for the matrix effect by adding morphine (10, 50, and 100 μg L^-1). The findings of the triplicate experiment showed that the relative recoveries of morphine and codeine in blood samples had a standard deviation of less than six and ranged from 91.8 to 107.0 %. The relative recoveries that were obtained indicated that the matrix unaffected the performance of the suggested method for extracting analytes from blood samples.

3.9 Comparison to other methods

Table 4 compares the analytical figures obtained from the pH-switchable DES-LPME technique with HPLC-UV for the preconcentration and identification of morphine and codeine in biosamples. Compared to other analytical procedures, the present method has a low LOD (0.5 μg/L) and a broader linear range (1.5–300 μg/L) with RSD < 5 %. The approach had a lower extraction time (<2 min) and a greater preconcentration factor (152 − 166) compared to other methods. The reported analytical methodology proved to be an effective, sensitive, and reliable method for analyzing pharmaceuticals in biological samples, as evidenced by the satisfactory results.