Recently, new psychoactive substances (NPSs) have emerged in illicit markets1,2. Although many countries are trying to curb this trend, e.g., more than 170 NPSs are currently controlled in China, NPSs still pose a threat to public health. Most NPSs are synthetic cannabinoids and designer cathinones, but there has been a sharp increase in the consumption of novel synthetic opioids, particularly fentanyl and its analogues3. Fentanyl, a μ-opioid receptor agonist, is used as an analgesic and anesthesia adjuvant with a 50–100 times higher potency than morphine4,5,6. However, numerous illicit fentanyl substances have been used to adulterate other abuse drugs such as heroin, cocaine, and methamphetamine. Fentanyl and its analogues, which have high potency and numerous side effects, e.g., respiratory depression, have caused many fatalities7. This problem is further aggravated by the easy availability of these substances via complex international networks and their use with other drugs6,8,9. Daniulaityte et al.10 reported that in Montgomery County (located in southeast Ohio), the number of unintentional overdose death cases in which positive tests for fentanyl analogues were obtained increased by 337% between the second half of 2015 and the first half of 2017.

As a consequence of increasing fatalities and the emergence of new fentanyl analogues and novel synthetic opioids, forensic laboratories must update their analytical methods for the identification and quantification of these drugs in various biological matrices. Some methods have been developed for the detection of these substances in whole blood11,12,13, urine14, saliva15,16,17, and dried blood spots18,19. However, it has been reported that the stability of some fentanyl analogues in whole blood is one month or less20,21. In other words, fentanyl analogues have a short period of storage time in such samples. Moreover, fentanyl is metabolized quickly in vivo, as the half-life of fentanyl in adults is 3.7 h and in infants is 5.3–21.1 h22. Therefore, to supplement these methods, it is necessary to develop methods for analyzing hair samples.

Hair, as an alternative or complementary matrix to conventional matrices, is a research hotspot in forensic science. Hair analysis can provide a longer detection window because substances may remain in hair for a long time without significant degradation. Furthermore, hair is easy to access, transport, and store23,24. To the best of our knowledge, only a few comprehensive methods using LC–MS/MS have been reported for analyzing fentanyl analogues and novel synthetic opioids in hair3,25. However, only a limited number of compounds were included in these methods.

Despite the advantages of hair analysis, the drug concentration in hair after a single use is usually on the pg/mg level26. Moreover, as fentanyl analogues typically have low active concentration27, higher sensitivity is required for the analysis of fentanyl analogues in hair. Compared with other methods, such as gas chromatography with nitrogen–phosphorous detection28 and gas chromatography–mass spectrometry29, liquid chromatography–tandem mass spectrometry30,31 is promising for analyzing fentanyl and its analogues in hair owing to its short analysis time, selectivity, and sensitivity.

In this study, an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the identification and quantification of 31 fentanyl analogues and 6 novel synthetic opioids in hair. Furthermore, the developed method was successfully applied to authentic cases.

Materials and methods

Chemicals and regents

Standards of fentanyl, norfentanyl, alfentanil, acetyl norfentanyl, U-47700, N-desmethyl U-47700, U-48800, U-50488, and W-18 (containing 1 mg/mL free base); 4-anilino-N-phenethylpiperidine (4-ANPP), norcarfentanil, acryl fentanyl, butyryl fentanyl, isobutyryl fentanyl, para/ortho-fluorofentanyl, para-fluorobutyryl fentanyl (PFBF), 4-fluoroisobutyryl fentanyl, cis-3-methylfentanyl, β-hydroxythiofentanyl, valeryl fentanyl, ocfentanil, furanyl fentanyl, sufentanil, remifentanil, carfentanil, cyclopropylfentanyl, and methoxyacetylfentanyl (containing 100 μg/mL free base); and acetyl fentanyl (containing 50 μg/mL free base) were obtained as methanol or acetonitrile solutions from CERILLIANT (Round Rock, TX, USA). 3-Methylthiofentanyl, trans-3-methylfentanyl, α-methylfentanyl, β-hydroxyfentanyl, β-hydroxy-3-methylfentanyl, thiofentanyl, and tetrahydrofuranyl fentanyl (THF-F) (1 mg/mL) were synthesized by the Criminal Investigation Department of the Shanghai Public Security Bureau (Shanghai, China). The deuterated internal standards (ISs) of fentanyl-d5 (used for most fentanyl analogues), norfentanyl-d5 (used for norfentanyl, acetyl norfentanyl, and norcarfentanil), and U-47700-d3 (used for the novel synthetic opioids) were purchased from Cerilliant.

High-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were obtained from SIGMA-SLDRICH (St. Louis, MO, USA). Formic acid (98%), ammonium acetate (98%), and ammonium formate (98%) were obtained from FLUKA (Seelze, Germany). Ultrapure water was produced using a Milli-Q system (MERCK, Darmstadt, Germany).

Solution preparation

Working solutions were obtained by diluting their single solutions in one methanol mixture, and further dilutions of this mixture in methanol. Working solutions were prepared at 10 different concentration levels (4, 10, 20, 100, 200, 400, 1,000, 2,000, 2,500, and 4,000 ng/mL).

An IS mixture (10 ng/mL) was obtained by spiking the extraction medium (EM), which consisted of methanol, acetonitrile, and 2 mmol/L ammonium formate (25:25:25, v/v/v, pH 5.3) with 250 μL of a mixture (100 ng/mL) of fentanyl-d5, norfentanyl-d5, and U-47700-d3 32.

Hair specimens

Blank hair samples, provided voluntarily by the laboratory staff, were used for spiked calibration standards and quality control (QC) samples. The real hair samples used were from suspected users who were arrested and investigated by police. All the samples were stored at room temperature until analysis. All participants provided written informed consent and all study protocols were approved by the Ethics Committee of Academy of Forensic Science, Shanghai, China.

Sample extraction

To remove contaminants, the samples were washed with water two times and acetone three times, and then air-dried at room temperature. The washed samples were cut into 2–3 mm pieces and weighed (20 mg) in 2 mL tubes. Then, ceramic beads and 1 mL of the EM (containing 10 ng/mL IS) were added to tube. Subsequently, the hair samples were extracted by cryogenic grinding using a Bead Ruptor system (OMNI, Kennesaw, GA, USA) at a speed of 6 m/s for 20 s and then allowed to cool for 40 s. This process was repeated 10 times. The pulverized samples were centrifuged for 3 min at 14,000 × g and filtered (pore size 0.22 μm). Finally, 200 μL of filtrate was transferred into an autosampler vial.

UHPLC-MS/MS conditions

The UHPLC-MS/MS analysis was performed on an Acquity UPLC system (Milford, MA, WATERS, USA) coupled to a QTRAP 6500 PLUS triple quadruple linear ion trap mass spectrometer (AB SCIEX, Framingham, MA, USA). Sample separation was performed using on a WATERS Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) fitted with a 1.8 μm HSS T3 guard column. The mobile phase was composed of 20 mmol/L ammonium acetate solution containing 0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B). The temperature of the autosampler was set at 4 °C and the injected volume was 5 μL. The gradient elution procedure is shown in Table 1.

Table 1 Steps of gradient elution.

The mass spectrometer was equipped with an electrospray interface operating in positive ionization mode. The source temperature and ion spray voltage were set to 500 °C and 5,500 V, respectively. The gas parameters were set as follows: collision-activated dissociation (CAD) gas, medium; curtain gas (CUR), 30 psi; nebulizing gas, 40 psi; and heater gas, 40 psi. Detection was performed using multiple reaction monitoring (MRM) with two transitions for each analyte and IS. The first transition was used for quantification and the second for qualification. The MRM transitions and optimized mass spectrometric parameters for each compound are listed in Table 2.

Table 2 MRM transitions and mass spectrometric parameters for analytes and internal standards.

Method validation

The method was developed according to the Society of Hair Testing (SoHT)33 guidelines and several recent criteria34,35,36 for method validation. Furthermore, the recovery and matrix effect (ME) were evaluated as described by Matuszewski et al.37.


The method selectivity was assessed using eight different sources of blank hair and spiking with the IS (10 ng/mL) to evaluate potential interference. Moreover, interference from possible coadministered medications was investigated according to our previous procedure38.

Limits of detections (LODs) and limits of quantification (LOQs)

To determine the LODs and LOQs, blank hair samples were spiked with analyte concentrations of 5.0, 2.5, 1.0, and 0.5 pg/mg, and three replicates of each concentration were analyzed. The concentration that gave a signal-to-noise (S/N) ratio greater than 3 for both the MRM transitions was chosen as the LOD. The LOQ was defined as the lowest calibration point with a coefficient of variation (CV) of less than 20% for the precision and accuracy in the range of 80–120%.

Calibration standards (2.0–2,500 pg/mg) were prepared by adding the working solutions to 20 mg of blank hair. In addition, QC samples were prepared at four concentration levels: LOQ (2 and 5 pg/mg), low (10 pg/mg), medium (500 pg/mg), and high (2,000 pg/mg). To determine linearity, seven sets of calibrators (two replicates for each set) were analyzed. The calibration curves were constructed by plotting the peak area ratios between each analyte and IS versus the concentration using 1/x weighting.

Accuracy and precision

The method precision and accuracy were assessed by analyzing spiked blank hair samples at four QC levels (LOQ, low, medium, and high). The precision was expressed as the CV. The intraday and interday precision were determined by analyzing six replicates on one day (n = 6) and over four days (n = 24), respectively. The CV for the precision should not exceed 15% for the low, medium, and high samples, whereas that for the LOQ sample should not exceed 20%. The accuracy was determined as the percentage ratio of the measured and theoretical values.

Recovery and ME

According to the method recommended by Matuszewski et al.37, the extraction recovery and ME were assessed at low (10 pg/mg), medium (500 pg/mg), and high (2000 pg/mg) levels. Hair samples from six drug-free individuals were used. For each level, the samples were divided into three groups (sets 1, 2, and 3). Set 1 consisted of neat standard solutions containing all the analytes in the EM. Set 2 was obtained by extracting the blank hair samples of six individuals and then spiking with the analytes. Set 3 was obtained by extracting the spiked hair samples using the method described in Sect. Sample extraction. The extraction recovery was calculated as the percentage ratio of the peak area of set 3 to the peak area of the set 2. The ME was defined as the percentage ratio of the peak area of set 2 to the peak area of set 1.


The stability of each analyte in hair was determined by injecting the extracted samples at three levels (n = 6) after storage in the autosampler at 4 °C for 24 h.

Ethics approval and consent to participate

The hair collection was carried out in accordance with SoHT guidelines. All participants provided written informed consent and all study protocols were approved by the Ethics Committee of Academy of Forensic Science, Shanghai, China.

Results and discussion

Method development

Chromatographic conditions

In general, screening methods for fentanyl analogues requires must address the separation of isomers, e.g., PFBF and 4-fluoroisobutyryl fentanyl. Fogarty et al.39 has reported a method for detecting 18 fentanyl analogues in whole blood and separating three pair of isomers (butyryl fentanyl and isobutyryl fentanyl, para-fluorofentanyl and ortho-fluorofentanyl, and β-methylfentanyl and α-methylfentanyl). In our study, 31 fentanyl analogues including 5 pairs of isomers were analyzed. To separate the isomers, we optimized the gradient elution based on previous studies13. First, we compared analyte separation using a WATERS T3 column and a RESTEK PPFP column (100 × 2.1 mm, 5 μm) and found that better separation was achieved using the former column (Fig. 1a).Furthermore, as the method for isomer separation in the previous study took a long time (30 min), the T3 column was still used13. Based on other previous reports39,40, we used methanol instead of acetonitrile and found that the solvent did not influence the separation of the chromatographic peaks significantly (Fig. 1b). With the previous method13, the gradient elution time was extended to achieve isomer separation. As shown in Fig. 1c, this method cannot separate the isomers. Following refinement of the gradient, the separation was still not ideal as shown in Fig. 1d. Therefore, we reduced the flow rate from 0.3 to 0.2 mL/min, which allowed separation of four pairs of isomers, but not para-fluorofentanyl and ortho-fluorofentanyl (Fig. 1e).

Figure 1
figure 1

Chromatograms under different liquid conditions. (a) Different columns-WATERS T3 column; (b) different organic mobile phase-acetonitrile; (c) extending analysis time; (d) modifying the gradient elution; (e) reducing the flow rate.

Sample extraction conditions

The sample preparation conditions were also optimized. Methanol is typically selected as the extraction solvent for hair samples in previously methods for quantifying fentanyl analogues3,41,42. However, the EM has also been used to extract analytes from hair samples32. Hence, methanol and the EM were compared as extraction solvents in our study. The chromatographic behavior of the compounds was better when EM was used as the extraction solvent, especially for the isomers.

Subsequently, extraction using different volumes (500, 800, and 1,000 μL) of the EM was investigated. The recoveries of all compounds were in the range of 84.34–96.08%, 89.10–108.63%, and 83.27–104.15% with volumes of 500, 800, and 1,000 µL, respectively, with ME values in the range of 358.40–504.59%, 79.49–115.06%, and 77.92–104.30%, respectively. The EM volume had a great impact on the ME. In particular, when the analytes were extracted with 500 μL of the EM, the ME value increased significantly. However, for the recovery, the effect of the EM volume was not significant. Finally, the extraction solution was EM and the volume was 1,000 μL.

Method validation


No interfering signals were observed at the retention times of the analytes and ISs. Furthermore, there was no interference in blank hair spiked with common drugs of abuse and pharmaceuticals. The retention times are summarized in Table 2 and the chromatograms of all the analytes in hair samples spiked at the LOQ concentration are shown in Fig. 2.

Figure 2
figure 2

MRM chromatograms for the 37 analytes in hair samples spiked at the LOQ concentration.

Linearity, LOD, and LOQ

Table 3 summarizes the LOD, LOQ, regression equation, and R2 value obtained for each analyte. To the best of our knowledge, this is the first method for quantifying 31 fentanyl analytes and 6 novel synthetic opioids. The LODs for all the compounds ranged from 0.5 to 2.5 pg/mg, and the LOQs ranged from 2 to 5 pg/mg. Busardò et al.25 reported a method to quantify 22 fentanyl analogues in hair with LODs of 3–7 pg/g and LOQs of 11–21 pg/g. The calibration curves of all the analytes were established in different concentration range, but with acceptable correlation coefficients (R2 > 0.99). According to previous reports3,25,41,43,44, the concentrations of fentanyl analogues in hair samples are typically in the following ranges: 3–2,800 pg/mg for fentanyl, 15.1–149 pg/mg for norfentanyl, and 3–104 pg/mg for 4-ANPP. In addition, a concentration of 44 pg/mg has been reported for furanyl fentanyl41. Therefore, the linearity ranges obtained for these compounds in our study cover the ranges observed in authentic cases.

Table 3 LODs, LOQs, and linearity for analytes in hair.

Precision and accuracy

The precision and accuracy obtained for each analyte are listed in Table 4. The intraday and interday precisions of the all compounds at the LOQ, low, medium, and high levels were less than 13.32%. Furthermore, the accuracies at the four levels ranged from 85.63% to 116.1%, except for acetyl norfentanyl at the LOQ, which had an accuracy of 116.1%. Thus, the precision and accuracy of the method are acceptable according to the previous criteria34,35,36.

Table 4 Precision and accuracy for analytes in hair.

Recovery and ME

The extraction recovery and ME data are summarized in Table 5. The recoveries of all the analytes from the QC samples at four levels ranged from 89.42 to 119.68%. The ME values were within the range of 85.76–119.77%, except for that of W-18, which was in the range of 44.81–54.11%. Chromatographic evaluation of this compound was less than optimal owing to large fluctuations during elution.

Table 5 Recovery and matrix effect for analytes in hair.


The stability results for each analyte are shown in Table 6. The stabilities at the three concentration levels were within the range of 77.44–113.71% for all the analytes after storage in the autosampler at 4 °C for 24 h. Therefore, the developed method is suitable for use in daily forensic work.

Table 6 Stability of analytes in hair.

Application to authentic cases

Following validation, the developed method was applied to the determination of fentanyl and its analogues in hair from authentic cases. The MRM chromatograms of cases 1 and 2 are shown in Fig. 3.

Figure 3
figure 3

Chromatograms of two MRM transition in the authentic cases.

Case 1

A 35-year-old female patient without a history of drug abuse underwent surgery for thyroid disease. Anesthesia induction was performed by endotracheal intubation during surgery. One month after the operation, the patient's hair was collected and then the 0–3 cm segment of the hair sample was analyzed. In this case, fentanyl was detected at a concentration of 8.02 pg/mg. Schneider et al.44.reported a case in which a patient with a chronic and heavy toothache was treated with a fentanyl patch for 22 consecutive days. For a 5 cm hair sample cut into segments 0–1 cm, 1–2 cm, 3–4 cm, and 4–5 cm, the fentanyl concentration in all the segments was in the range of 60 pg/mg (LOQ) to 480 pg/mg. Compared with multiple doses, the concentration of fentanyl in hair was lower after a single dose, which may be why norfentanyl was not detected in our real case.

Case 2

A 51-year-old man was reported to police by his colleague. According to the informant, the man may have been using drugs for a long time. After hair collection, the hair sample was cut into three segments (S1: 0–3 cm, S2: 3–6 cm, and S3: 6–9 cm). Then, these samples were analyzed using our proposed method. Sufentanil was detected in the hair sample at concentrations of 183.91, 131.68, and 31.48 pg/mg for S1, S2, and S3, respectively. However, no metabolites were detected in the hair sample owing to the parent drugs being largely incorporated inside the keratin matrix from sweat, the bloodstream, and the sebum before metabolization. In this case, the observed concentration of sufentanil in hair will provide a reference for future forensic work.


In this study, a sensitive, simple, rapid, and robust UHPLC-MS/MS was developed and validated for determination of 31 fentanyl analogues and 6 novel synthetic opioids in hair samples. This method covers fentanyl analogues and novel synthetic opioids that are common or new to the drug market. Furthermore, the developed method was successfully applied to authentic cases.