ABSTRACT
Objective
2D-LC method and LC-MS/MS self-calibrated internal standard method were used to compared the consistency and correlation of quetiapineserum concentration.
Methods
Quality control samples at low, medium, and high concentrations of quetiapine, as well as serum samples from 138 psychiatric patients taking quetiapine, were analyzed using 2D-LC and LC-MS/MS self-calibrated internal standard method, respectively. Performed a complete validation of both methods. The Bland–Altman plot was used to assess the consistency of the results, while Pearson correlation analysis and Passing–Bablok regression were used to analyze the correlation among the results.
Outcomes
The linear range for 2D-LC was 60.74–1518.48 ng/ml, and the linear range for LC-MS/MS self-calibrated internal standard method was 60.88–1522.02 ng/ml. For both methods, the relative standard deviation of intraday and daily precision was less than 15%. Calculated using the Bland–Altman plot, the 95.65% LoA was (−11.85, 30.82). The Passing–Bablok regression equation was OLZLC-MS/MS = 2.44 + 1.06 × OLZ2D-LC, and the Pearson correlation coefficient was 0.994 (p < 0.001).
Conclusion
The 2D-LC and LC-MS/MS self-calibrated internal standard method, exhibit exceptional agreement and correlation in the quantification of quetiapine concentrations in serum. which can be used for conventional therapeutic drug monitoring (TDM).
KEYWORDS: 2D-LC, LC-MS/MS, self-calibrated internal standard method, quetiapine, serum concentration
1. Introduction
Quetiapine is a second-generation antipsychotic medication that is employed to treat schizophrenia and bipolar disorder(BD), as well as as an adjunctive treatment for major depressive disorder(MDD) [1]. It is well-tolerated and provides an excellent alternative for individuals who are hypersensitive to medications like clozapine or olanzapine. According to reports, the suicide rate among patients with MDD is 20 times higher than that of the general population, while the suicide rate among patients with BD is approximately 20–30 times higher [2,3]. Research by Ke Wang et al. indicates that treating patients with MDD and BD using a combination of cognitive behavioral therapy and quetiapine can effectively reduce the risk of suicide, improve depressive symptoms, and enhance coping abilities in adults with mood disorders [4]. Research has demonstrated that quetiapine’s effects on schizophrenia may be mediated by the blockade of dopamine receptor and serotonin 2A receptor transmission, while its effects on bipolar depression and major depressive disorder may be mediated by the drug or its metabolites binding to norepinephrine transporters [5]. Nevertheless, certain patients may experience adverse reactions when taking quetiapine, including somnolence, orthostatic hypotension [5], elevated serum lipids [6], or even toxic coma [7]. This is why it is essential to develop a rapid method for detecting quetiapine serum concentrations. At present, the primary methods for detecting quetiapine serum concentrations are liquid chromatography-tandem mass spectrometry (LC-MS/MS) [8–10], high-performance liquid chromatography with diode array detection (HPLC-DAD) [11], high-performance liquid chromatography with ultraviolet detection (HPLC-UV) [12,13], and capillary electrophoresis [14]. At present, there are no Class II reagents available in China for the detection of psychotropic substances using liquid chromatography and mass spectrometry. There are no unified standards, and the majority of Chinese laboratories have devised their own methods using Class I reagents. Two-dimensional liquid chromatography (2D-LC) has a reduced detection speed but lower detection costs. It is capable of purifying and separating target components in addition to measuring their concentration. LC-MS/MS is capable of determining the molecular weight of target compounds and exhibiting high sensitivity and specificity. However, it is limited to the analysis of samples and is unable to purify them. Each technique has its own set of advantages and disadvantages. We assume that there is a degree of agreement and discrepancy between the 2D-LC method and the LC-MS/MS method. Therefore, we determined the serum concentrations of quetiapine using both methods to investigate whether they are consistent or show discrepancies, thereby providing a reference for the selection of analytical methods for the clinical monitoring of quetiapine serum concentrations.
2. Materials and methods
2.1. Materials
2.1.1. Instruments
Hunan Demite Instrument Co., Ltd. FLC2701 SPD-M30A Fully Automatic Two-Dimensional Liquid Chromatography System; Hunan Demite Instrument Co., Ltd. LC/MS, Ms-Mate9500 High-Performance Liquid Chromatography Tandem Mass Spectrometry Detection System; Sichuan Shuke Instrument Co., Ltd. TGL-2150 High-Speed Freezing Centrifuge.
2.1.2. Reagent
Calibration standards and control materials obtained from Hunan Demite Instrument Co., Ltd. Calibration standard batch number: 2D-LC :Calibration standard lot number: 20220719; Quality control standard lot number: 20220110.
LC-MS/MS:Calibration standard lot number: 20221219; Quality control standard lot number: 20230221.
2.2. Sample collection
Serum samples were collected 8 hours after the administration of quetiapine from 138 patients with mental disorders who received treatment at Qujing Third People’s Hospital between May 2023 and June 2024. These samples were used for a comparison of the consistency between the 2D-LC and LC-MS/MS methods.
Serum samples were collected from eight distinct sources, including jaundiced patients, hyperlipidemic patients, outpatient physical examination subjects, patients with elevated transaminases, patients with hyperuricemia, hemolytic specimens, and individuals taking clozapine. Each source was represented by one patient from Qujing Third People’s Hospital, horse serum is sourced from Hunan Demite Instrument Co., Ltd, with all samples serving as matrix-validated research specimens.
Centrifuge the collected samples at 3000 rpm for 5 minutes to separate the serum, which should be set aside for later use.
All clinical samples involved in this study have been approved by the Ethics Committee of Qujing Third People’s Hospital.
2.3. 2D-LC measurement conditions and serum sample processing
2.3.1. Chromatographic conditions
Chromatographic conditions are as follows: Chromatographic column: One-dimensional column: SXI 3.5*25 mm *5 μm; Intermediate column: SCB 3.0 *10 mm *2.8 μm; Two-dimensional column: MSCB 3.0*50 mm *3.3 μm; Reagents: One-dimensional reagents: CAA-1D mobile phase type CAA-1B; Two-dimensional reagents: BPI3-2A: API3-2A: MPI-2A: OPI-2A = 22:37:30:11; Auxiliary reagents: Pure water. Processing method: ORG-1 processing temperature: 50°C; Detection wavelength: CH1: 296 nm; Injection volume: 100 μL; CH2: 250 nm; Method name: Quetiapine-Secondary Single-Channel Method-100 μL/min; Flow rate: Pump A: 0.6 mL/min; Pump B: 0.01 mL/min; Pump C: 0.7 mL/min; Time program: Secondary single-channel constant-pressure pump stops automatically.
2.3.2. Detection principle
The two-dimensional liquid chromatography system integrates functional modules such as a one-dimensional column (LC1), a two-dimensional column (LC2), a detector, and an autosampler. First, LC1 is used to perform a preliminary separation of serum samples, achieving baseline separation of quetiapine from most endogenous interferences such as proteins, lipids, and small-molecule metabolites. Subsequently, the target fraction is precisely eluted and transferred via an intermediate column to LC2 for further refined separation, effectively subjecting the target analyte to dual purification and focusing. Finally, the sample is analyzed using a UV detector.
2.3.3. Serum sample processing
Using a 1.5-mL PE centrifuge tube, draw 400 μL of serum, add 1,000 μL of ORG-1 protein removal reagent to the supernatant serum, vortex for 1 minute, centrifuge at 14,500 rpm for 8 minutes, and collect the supernatant for isocratic mode analysis via 2D-LC.
2.4. LC-MS/MS self-calibration method measurement conditions and serum sample processing
2.4.1. Chromatographic conditions
Chromatography column: MSCB-2A 3.0 * 50 mm *2.5 μm (dual columns in alternation); Quetiapine mass-to-charge ratio (M/Z): 384; Sample preparation: 200 μL sample +500 μL MARG-1, nebulizer flow rate: 1.5 L/min, characteristic ion: 384+, heating block temperature: 350°C, flow rate: 0.4 mL/min, drying gas flow rate: 10 L/min, temperature: 45°C, Qarray RF: 0 V, injection volume: 1 μL, ion source mode: ESI+.
2.4.2. Detection principle
The LC-MS/MS self-calibrated internal standard method utilizes quetiapine itself as the internal standard. Using the Ms-Mate9500 high-performance liquid chromatography-tandem mass spectrometry detection system, which is equipped with a mass spectrometry coupling system, the timing of sample and internal standard entry into the mass spectrometer is controlled via a time program. The sample to be tested is introduced first, followed by the internal standard 0.35 minutes later. Under identical chromatographic and mass spectrometric conditions, the differing retention times of the components allow the internal standard and the target compound to be separated. A standard curve is plotted with the concentration of the quetiapine standard on the x-axis and the ratio of the peak areas of quetiapine to the internal standard quetiapine on the y-axis, from which the concentration of the quetiapine sample under analysis is calculated.
2.4.3. Serum sample processing
Transfered 200 μL of serum into a 1.5 mL PE centrifuge tube, added 500 μL of MARG-1-pretreated supernatant serum (deproteinised), vortex for 1 minute, centrifuge at 14,500 rpm for 8 minutes, and used the supernatant for isocratic mode analysis via LC-MS/MS self-calibration.
2.5. Methodological examination
Validation was carried out in accordance with the ‘Guidelines for the Validation of Quantitative Analytical Methods for Biological Samples’ (9012) of the Chinese Pharmacopoeia [15] and the ‘Guidance for the Validation of Analytical Methods for Biological Samples’ issued by the US Food and Drug Administration (FDA) [16]. The validation process included specificity, standard curve, limit of quantification, accuracy and precision, extraction recovery, and stability.
2.6. 2D-LC
2.6.1. Exclusivity
After administering quetiapine, collected patient serum samples, blank serum from healthy people, and quetiapine calibration standards. Then, proceed as described in “2.3 Serum Sample Processing” to extract the supernatant for analysis.
2.6.2. Standard curve and quantitative limit
Prepared drug-containing Calibration serum solutions with concentrations of 60.74, 151.85, 303.70, 607.39, 911.09, and 1518.48 ng/mL. Followed the procedure in “2.3 Serum Sample Preparation.” Performed linear regression analysis using 2D-LC, plotting the peak area of quetiapine (Y) against mass concentration (X). The coefficient of correlation (R2) for the standard curve should be ≥0.99, and the limit of quantification should satisfy a signal-to-noise ratio (S/N) of ≥10.
2.6.3. Precision and recovery rate
Prepare six samples each of low (166.85 ng/mL), medium (667.40 ng/mL), and high (1334.79 ng/mL) concentrations of quetiapine serum samples. Six replicates should be carried out for each concentration and calculate the intra-day and inter-day precision.The relative standard deviation (RSD%) should be ≤15%.
2.6.4. Matrix validation
Taked serum from eight different sources as the blank serum. Adhere to the protocol outlined in “2.3 Serum Sample Preparation.” Introduced the supernatant to serum samples at three concentrations: 166.85, 667.40, and 1334.79 ng/mL. Proceed with injection for analysis, measured each concentration three times, and acquired peak regions.The ratio of the peak area to that of the corresponding quetiapine standard solution at the same concentration was the matrix effect result [9,17]. RSD% should be ≤15%.
2.6.5. Stability
Prepare low (165.01 ng/mL), medium (630.01 ng/mL), and high (1230.01 ng/mL) concentrations of drug-containing serum, and examine their stability by placing them at room temperature for 6 hours and repeatedly freezing and thawing them three times at −20°C. Measured each concentration three times. RSD% should be ≤ 15%
2.7. In-house calibration internal standard method of LC-MS/MS
2.7.1. Exclusivity
Taked blank serum from healthy individuals and added quetiapine internal standard, taked reference standard and added internal standard, and taked serum samples from patients after administration of the drug. Follow the procedure in “2.4 Serum Sample Processing,” take the supernatant, and analyze it.
2.7.2. Standard curve and quantitative limit
Prepare drug-containing serum solutions with concentrations of 60.88, 152.20, 304.40, 608.81, 913.21, and 1522.02 ng/mL. Follow the procedure in “2.4 Serum Sample Preparation” to perform linear regression between the area ratio (Y) of the target and internal standard and the mass concentration (X).The coefficient of correlation (R2) for the standard curve should be ≥ 0.99, and the limit of quantification should satisfy a signal-to-noise ratio (S/N) of ≥20.
2.7.3. Precision and recovery rate
Prepare six samples each of low (151.00 ng/mL), medium (604.01 ng/mL), and high (1208.01 ng/mL) concentrations of quetiapine serum samples.Six replicates should be carried out for each concentration and calculated the intra-day and inter-day precision. RSD% should be ≤15%.
2.7.4. Matrix validation
Take serum from eight different sources and water as the blank serum. Adhere to the protocol outlined in “2.4 Serum Sample Preparation.” Introduced the supernatant to serum samples at three mass concentrations: 151.00, 604.01, and 1208.01 ng/mL. For each sample analysis, performed three replicative measurements at each concentration, and calculate the matrix effect [9,17] and the matrix factor(MF) normalized to the internal standard.
2.7.5. Stability
Prepare low (120.01 ng/mL), medium (537.01 ng/mL), and high (1314.01 ng/mL) concentrations of drug-containing serum, and place them at room temperature for 6 hours, then freeze and thaw them three times at −20°C to examine their stability. Measured each concentration three times. RSD% should be ≤15%
2.8. Comparing the consistency of the two methods
After centrifugation, a total of 138 quetiapine blood samples were divided into two portions and analyzed using two different methods. The results of the two methods were compared for consistency.
2.9. Statistical processing
This study was based on fundamental statistical principles and employed descriptive methods to calculate the mean ± standard deviation ( ± s) and evaluate the relative standard deviation (RSD%). GraphPad 10.6 software was used to plot Bland–Altman plots to assess the agreement between the two methods, whilst R was used to perform Passing–Bablok regression and Pearson correlation analyses to evaluate the correlation between the two methods. A p-value of <0.05 was considered statistically significant.
3. Result
3.1. 2D-LC
3.1.1. Exclusivity
The retention time of quetiapine is approximately 4.37 mins. After administration, the retention time of quetiapine in serum is consistent with that of the standard sample, indicating good method specificity (Figure 1(A-C)).
Figure 1.
Examination of the specificity of 2D-LC.
(A): Blank serum; (B): Quetiapine calibration standard (the peak on the right is the target peak); (C): Serum after the patient took quetiapine.
Note: The calibration standard analyzed in Figure 1(B) is a multi-component standard containing several drugs. The peak on the left corresponds to other substances and is not relevant to this experiment. The target peak in this experiment was identified by analyzing a calibration solution (a methanol solution containing only quetiapine) and confirming that its retention time matched that of the target peak.
3.1.2. Standard curve and quantitative limit
The linear equation for quetiapine is: Y = 338.76X − 890.67,R2 = 0.9999 (n = 6). The linear relationship is good within the range of 60.74–1518.48 ng/mL, with a lower limit of quantification of 60.74 ng/mL.
3.1.3. Precision and recovery rate
The intra-day and inter-day RSD were both less than 15%; accuracy fell within the range of 85%–115%; the method recovery ranged from 95.43% to 100.01%; and the extraction recovery ranged from 97.25% to 99.33% (Table 1).
Table 1.
Determination of precision and recovery rate of quetiapine by 2D-LC.
| Concentration (ng/mL) | Intra-day (ng/mL) |
RSD (%) | Accuracy (%) | Inter-day (ng/mL) | RSD (%) | Accuracy (%) | Relative recovery (%) | Extraction recovery (%) |
|---|---|---|---|---|---|---|---|---|
| 166.85 | 166.53 ± 0.72 | 0.43 | 99.81 | 165.48 ± 4.52 | 2.73 | 99.18 | 99.18 | 98.80 |
| 667.40 | 667.03 ± 1.32 | 0.20 | 99.94 | 636.91 ± 24.51 | 3.85 | 95.43 | 95.43 | 99.33 |
| 1334.79 | 1334.80 ± 3.24 | 0.24 | 100.00 | 1334.87 ± 27.30 | 2.05 | 100.01 | 100.01 | 97.25 |
The data presented are mean ± SD or(percentage), RSD: standard deviation.
3.1.4. Matrix validation
The RSD for quetiapine at low, medium and high concentrations was less than 15%. The 2D-LC detection method utilizes the dual separation of interfering substances via a one-dimensional and a two-dimensional column, which significantly reduces interference from matrix effects. Furthermore, as the analysis is performed using non-destructive detection by a UV detector, peak areas remain constant regardless of the number of measurements; consequently, interference from the plasma matrix is minimal (Supplementary Table S1).
3.1.5. Stability
The mean values of the measured results for low, medium and high concentrations of quetiapine deviated from the theoretical concentrations by no more than ±15% (Table 2).
Table 2.
2D-LC stability test results.
| Testing time | Concentration (ng/mL) | Actual average (ng/mL) | RSD (%) | Accuracy (%) |
|---|---|---|---|---|
| At room temperature for 6 hours | 165.01 | 159.09 ± 6.10 | 3.83 | 96.41 |
| 630.01 | 622.03 ± 9.81 | 1.58 | 98.73 | |
| 1230.01 | 1221.92 ± 11.51 | 0.94 | 99.34 | |
| −20°C repeated freeze-thaw cycles 3 times | 165.01 | 158.21 ± 4.84 | 3.06 | 95.88 |
| 630.01 | 616.57 ± 8.61 | 1.40 | 97.87 | |
| 1230.01 | 1218.95 ± 12.50 | 1.03 | 99.10 |
The data presented are mean ± SD or(percentage), RSD: standard deviation.
3.2. In-house calibration internal standard method of LC-MS/MS
3.2.1. Exclusivity
The retention time of quetiapine is roughly 2.5 minutes. The serum retention period of quetiapine post-administration aligns with that of the standard sample, indicating strong technique specificity (Figure 2(A-C)).
Figure 2.
Examination of the specificity of the LC-MS/MS self-calibrated internal standard method.
(A): Blank serum with internal standard; (B): Quetiapine calibration standard with internal standard (left peak is the target peak); (C): Patient serum sample after taking quetiapine (left peak is the target peak).
Note: The target peak is identified by analyzing a calibration solution (a methanol solution containing only quetiapine) and confirming that the retention time of the calibration solution matches that of the target peak.
3.2.2. Standard curve and quantitative limit
The linear equation for quetiapine is: Y = 0.0032X − 0.0028, R2 = 0.9996 (n = 6). The linear relationship is good within the range of 60.88–1522.02 ng/mL, with a lower limit of quantification of 60.88 ng/mL.
3.2.3. Precision and recovery rate
The intra-day and inter-day standard deviations were both less than 15%; accuracy fell within the range of 85%–115%; the method recovery ranged from 99.35% to 100.37%; and the extraction recovery ranged from 94.70% to 96.30% (Table 3).
Table 3.
Determination of precision and recovery of quetiapine by LC-MS/MS self-calibrated internal standard method.
| Concentration (ng/ml) | Intra-day (ng/mL) |
RSD (%) | Accuracy (%) | Inter-day (ng/mL) | RSD (%) | Accuracy (%) | Relative recovery (%) | Extraction recovery (%) |
|---|---|---|---|---|---|---|---|---|
| 151.00 | 150.79 ± 2.66 | 1.76 | 99.86 | 150.02 ± 5.98 | 3.99 | 99.35 | 99.35 | 94.70 |
| 604.01 | 604.80 ± 4.82 | 0.80 | 100.13 | 606.26 ± 16.28 | 2.69 | 100.37 | 100.37 | 94.89 |
| 1208.01 | 1208.89 ± 9.69 | 0.80 | 100.07 | 1208.33 ± 21.6 | 1.79 | 100.03 | 100.03 | 96.30 |
The data presented are mean ± SD or(percentage), RSD: standard deviation.
3.2.4. Matrix validation
The standard deviation for quetiapine at low, medium and high concentrations was less than 15% in all cases, with the recovery (MF) falling within the range of 85% to 115%. The LC-MS/MS method using an internal standard employs quetiapine as the internal standard. As the target compound and the internal standard are the same substance with identical physicochemical properties, it is ensured that there is no difference between the two under the same mass spectrometry conditions. By calculating the corresponding ratio of the internal standard to the analyte, the common matrix effect affecting both can be offset, thereby minimizing plasma matrix interference (Supplementary Table S2).
3.2.5. Stability
The mean values of the measured results for low, medium and high concentrations of quetiapine deviated from the theoretical concentrations by no more than ±15% (Table 4).
Table 4.
Stability test results of the LC-MS/MS self-calibrated internal standard method.
| Testing time | Concentration (ng/mL) | Actual average (ng/mL) | RSD (%) | Accuracy (%) |
|---|---|---|---|---|
| At room temperature for 6 hours | 120.02 | 119.67 ± 3.61 | 3.01 | 99.71 |
| 537.01 | 527.82 ± 5.78 | 1.09 | 98.29 | |
| 1314.01 | 1308.02 ± 9.47 | 0.72 | 99.54 | |
| −20°C repeated freeze-thaw cycles 3 times | 120.02 | 116.89 ± 3.46 | 2.96 | 97.39 |
| 537.01 | 526.62 ± 6.36 | 1.21 | 98.07 | |
| 1314.01 | 1282.13 ± 12.22 | 0.95 | 97.57 |
The data presented are mean ± SD or(percentage), RSD: standard deviation.
3.3. Consistency comparison and correlation analysis
Calculated using the Bland–Altman method, 95.65% of the differences fell within the limits of agreement; therefore, the 2D-LC and LC-MS/MS methods using internal standards can be considered to show good agreement. The Bland–Altman plot is shown in Figure 3. The Passing–Bablok regression method yielded the equation: OLZLC-MS/MS = 2.44 + 1.06×OLZ2D-LC. The slope of the equation was 1.06 (95% CI = 1.03–1.08), and the intercept was 2.44 (95% CI = 0.089–6.52). Pearson correlation analysis yielded R = 0.994 (p < 0.001) (Table 5, Figure 4), indicating a strong correlation between the 2D-LC and LC-MS/MS self-calibration internal standard methods.
Figure 3.
Bland–Altman bias plot of quetiapine serum concentrations.
Table 5.
Comparison of consistency between 2D-LC and LC-MS/MS self-calibrated internal standard method.
| 2D-LC (n = 138) | LC-MS/MS self-calibrated internal standard method (n = 138) |
R | p value | |
|---|---|---|---|---|
| Quetiapine concentration (ng/mL) | 99.06 ± 91.06 | 108.55 ± 95.99 | 0.994 | <0.001 |
Figure 4.
Correlation between 2D-LC and LC-MS/MS self-calibrated internal standard method detection values.
3.4. Clinical applications
According to a consensus published in 2018 by the Therapeutic Drug Monitoring (TDM) Expert Group of the Society of Neuropsychopharmacology and Psychopharmacology, monitoring quetiapine serum concentrations is considered to have clinical significance in improving the rational use of medication. Therefore, the clinical implementation of quetiapine TDM is recommended, with the recommended therapeutic range being 100–500 ng/mL [18]. Some hospitals also conduct routine serum concentration monitoring for quetiapine and have reported that, in clinical patients taking 150–750 mg/d of quetiapine, blood concentrations generally fall within the range of 0–600 ng/mL [19].
3.4.1. Case 1
A 27-year-old female patient was admitted on 20 June 2024 with a provisional diagnosis of ‘bipolar disorder’ following a history of ‘intermittent mood instability for over three years.’ She was initially prescribed ‘0.5 g/day of sustained-release magnesium valproate and 200 mg/day of quetiapine fumarate’ for mood stabilization and antipsychotic treatment. On 17 July, the blood concentration of quetiapine was measured at 78.67 ng/mL. The patient’s symptoms had improved. To ensure medication safety and minimize the occurrence of adverse drug reactions, the current treatment regimen was continued. On 12 September, during a clinical ward round, the consultant noted that the patient was agitated and emotionally unstable, and instructed that the dose of quetiapine be increased to 500 mg daily. The patient’s mood improved markedly compared to previously, with no reports of specific discomfort. A follow-up quinolizine plasma concentration test on 28 September showed a value of 185.24 ng/mL. The patient’s condition was largely under control, and the relevant treatment and TDM were continued. The use of 2D-LC for quinolizine TDM not only assists clinicians in optimizing dosing regimens and enhancing therapeutic efficacy but also provides an objective basis for individualized dose adjustments.
3.4.2. Case 2
The patient, a 59-year-old woman, presented with a history of “talking to herself, delusions of persecution and disorganised behaviour” spanning 24 years, with symptoms having worsened over the past five years. On 10 August 2024, she was admitted with a provisional diagnosis of ‘paranoid schizophrenia’ and treated with ‘200 mg of quetiapine tablets every evening; 10 mg of aripiprazole tablets daily’ and other medications. The patient’s serum total bilirubin was 15.5 μmol/L, serum direct bilirubin was 2.0 μmol/L, serum alanine aminotransferase (ALT) 11 U/L, serum aspartate aminotransferase (AST) 6 U/L, and creatinine 44 μmol/L. The clinician recommended a quetiapine therapeutic drug monitoring (TDM) test. Following the test, the quetiapine TDM result was 561.76 ng/mL. The patient continued to take quetiapine and other medications, with regular TDM monitoring. On 24 September, quetiapine TDM was re-checked, with a concentration of 1517.49 ng/mL, reaching the clinically critical range. Liver function tests showed ALT 44 U/L, AST 49 U/L, indicating liver damage. As the patient reported no discomfort, the dosage of ‘Quetiapine 200 mg tablets every evening’ was adjusted to ‘100 mg every evening’ and administration was suspended that night. The following day, the quetiapine TDM concentration was re-measured at 646.49 ng/mL, a significant decrease from the previous level. The patient was advised to temporarily maintain the current medication regimen. Rapid and accurate determination of the patient’s quetiapine TDM levels via LC-MS/MS using an in-house standard method effectively reduced the risk of adverse drug reactions.
4. Discussion
In recent years, ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) has become the method of choice for population pharmacokinetic studies due to its exceptional stability, sensitivity and high throughput [20–22]. However, as this technique requires expensive equipment, specialist maintenance and strict environmental control, it is not suitable for routine testing in practical applications. HPLC is one of the most commonly used instrumental analytical methods for addressing drug research challenges in both academia and industry; for the detection of quetiapine, HPLC is currently the most mature and extensively validated method [5]. Due to the limitations in detection sensitivity of HPLC-UV, sample preparation often requires liquid-liquid extraction and concentration methods, which increase the time and complexity of the process. Consequently, it is difficult to achieve rapid and accurate monitoring of quetiapine serum concentrations in patients with poisoning [9]. Furthermore, the HPLC-DAD method can only analyze compounds that absorb ultraviolet light, and its sensitivity is consistently inferior to that of LC-MS/MS. Due to its advantages of high specificity, high resolution and high analytical efficiency, with detection limits reaching the picogram level, LC-MS/MS is the method of choice for trace analysis in complex matrices, such as TDM. Consequently, it is increasingly being applied in clinical TDM [23–26].
In LC-MS/MS analytical methods, internal standards for quetiapine include isotopes and aripiprazole [9]. Among these, isotopically labeled compounds, when used as internal standards, do not react with quetiapine and exhibit virtually identical chromatographic retention and mass spectrometric fragmentation patterns to the analyte; they are also relatively stable, making them the most commonly selected internal standards at present [23–25]. However, they are relatively expensive, difficult to obtain and involve high analytical costs, which hinders the implementation of routine monitoring.The LC-MS/MS self-calibrating internal standard method [27] employed in this study utilizes quetiapine itself as the internal standard. Although it is the same substance as the quetiapine sample under analysis, the timing of the internal standard’s entry into the chromatographic column is controlled by a self-calibrating internal standard pump to differ from that of the sample. This time difference separates the two, resulting in two distinct peak areas; this ensures both that their structures are identical and that the results do not interfere with one another. Utilizing the relationship whereby the area ratio of the internal standard to the target is directly proportional to concentration, the concentration of the quetiapine sample under analysis is calculated via a calibration curve, thereby serving as an alternative to the LC-MS/MS isotope ratio method. At the same time, this method reduces the risk of detection failure caused by the loss of internal standards due to improper handling during sample preparation. Furthermore, as the sample injection volume is only 1 µl, it reduces quality control costs, and the time required to analyze a single sample is approximately 3 minutes, significantly shortening the testing time and effectively achieving the objectives of economical, rapid and efficient analysis.The current limitations of the LC-MS/MS method lie in the fact that it imposes rather stringent requirements on the experimental environment; the mass spectrometry system is highly sensitive to fluctuations in environmental factors such as voltage, temperature and humidity, and requires stable laboratory conditions to ensure the normal operation of the instrument and the reliability of the results. This undoubtedly increases the difficulty of daily laboratory maintenance and management, whilst also posing a risk of ion suppression or enhancement effects, thereby affecting the accuracy of quantification. Furthermore, the method places high demands on the technical proficiency of the operators, which limits its widespread adoption.
Compared with conventional LC-MS/MS methods, 2D-LC offers comparable quantitative performance whilst providing simpler operation, lower running costs and minimal maintenance, making it a practical alternative for hospitals and clinical laboratories. It simplifies sample preparation, reduces operational costs and avoids common issues such as ion suppression; consequently, an increasing number of hospitals are adopting 2D-LC platforms for the measurement of serum drug concentrations of various medications [28–31]. The 2D-LC technique established in our laboratory [32] for quetiapine detection necessitates merely 100 μL of serum sample volume, thereby considerably minimizing sample usage. The quantity of control samples necessary for concurrent testing is significantly diminished, therefore reducing control expenses. The procedure is straightforward and rapid, with each sample analysis requiring roughly 5.5 minutes, rendering it more efficient and sensitive than traditional liquid chromatography techniques employed at academic institutions. The main limitations of 2D-LC are that it is not suitable for high-throughput analysis; it has poor mobile phase compatibility, making it prone to peak distortion and baseline drift; and method development is challenging, with a lengthy optimization period.
The study’s results indicated that both approaches exhibited high precision, recovery rates, and stability, with no matrix interference seen. The detection results for clinical samples demonstrated strong consistency and correlation, yielding dependable outcomes. When comparing the two methods, the LC-MS/MS method using an internal standard demonstrates higher sensitivity and specificity; Its multiple reaction monitoring mode enables precise differentiation between quetiapine and its structurally similar metabolites, avoiding potential peak overlap interference in UV detectors. It supports rapid analysis within 3–5 minutes and can detect quetiapine quickly and accurately; however, it is relatively expensive and is more suitable for the precise, high-throughput quantitative requirements of large-scale samples analysis in clinical therapeutic drug monitoring. In the detection process, 2D-LC primarily utilizes a UV detector; it does not require internal standards, and the mobile phase used is chromatographically pure. It is relatively inexpensive and simple to operate, but its detection speed is slower than that of the self-calibrating internal standard method used in LC-MS/MS. It is therefore suitable for laboratories with a small number of samples. Regarding drug component analysis, 2D-LC effectively separates and purifies target components, rendering it appropriate for scientific investigation. The LC-MS/MS self-calibrated internal standard method is exclusively applicable for sample detection and is incapable of separating or purifying samples; nonetheless, it can determine the molecular weight and structure of compounds. Each strategy possesses distinct advantages and disadvantages.
5. Conclusion
This study demonstrates that both LC-MS/MS self-calibrated internal standard method and 2D-LC exhibit good precision, recovery rates, and stability in detecting quetiapine serum concentrations, with no matrix interference, enabling timely provision of TDM testing services in clinical settings. Among these, LC-MS/MS self-calibrated internal standard method offers higher sensitivity and specificity with rapid detection speed, making it suitable for laboratories handling a large of samples. Conversely, 2D-LC features simple operation, accurate results, and lower costs, making it ideal for laboratories with smaller samples.
Supplementary Material
Funding Statement
This paper received no funding.
Article highlights
A rapid, sensitive and cost-effective 2D-LC method has been established for the quantitative determination of quetiapine in human serum.
A sensitive and highly selective LC-MS/MS method has been established for the quantitative determination of quetiapine in human serum.
This LC-MS/MS self-calibrated internal standard method uses quetiapine itself as an internal standard, offering the advantages of an isotope-based internal standard whilst being economical and rapid; it can serve as an alternative to isotope-based LC-MS/MS methods.
Simultaneous determination of quetiapine using 2D-LC and LC-MS/MS methods: a comparison of the agreement and differences between the two methods.
LC-MS/MS and 2D-LC exhibit good precision, recovery rates, and stability in detecting quetiapine serum concentrations, enabling timely provision of TDM services in clinical settings.
Disclosure statement
Both analytical methods described in this paper are in-house methods based on Class I reagents. The author, Yan Xuefang, has registered patents in Luxembourg, Europe, and obtained patent certificates. Specifically: the patent for a mass spectrometry method with an internal standard for determining the serum concentration of quetiapine is numbered LU50789; the patent for a two-dimensional liquid chromatography method for determining the serum concentration of quetiapine is numbered LU601252.
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony,grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships disclose.
Ethical declaration
All our research protocols and data collection were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Qujing Third People’s Hospital. As our study was retrospective in nature, informed consent forms were not obtained from patients. The Ethics Committee of Qujing Third People’s Hospital waived the requirement for informed consent.
Data availability statement
The authors confirm that the data supporting the findings of this study are available in the article and its supplementary materials.
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/17576180.2026.2668682
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Supplementary Materials
Data Availability Statement
The authors confirm that the data supporting the findings of this study are available in the article and its supplementary materials.