Quantitative analysis of hair samples for 1-benzylpiperazine (BZP) using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) detection

Technical Note


Benzylpiperazine (BZP) is an amphetamine-type stimulant, which was legally available in New Zealand and widely used in “Party Pills” until reclassification as a Class C drug in April 2008. BZP was included as part of a multi-analyte method developed for hair screening using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS). A 20-mg sample of hair is extracted and partially purified using mixed-mode solid-phase extraction cartridges prior to analysis by LC-MS/MS. The method was developed as a broad screen for drugs of abuse (including amphetamines, opiates, and benzodiazepines), with only the BZP results being presented here. The assay was validated and found to be linear over the range of 0.085 to 8.65 ng/mg with correlation coefficient of r2 ≥ 0.99. Blank hair samples spiked with BZP at 0.22 and 2.16 ng/mg gave intra- and inter-day precision coefficients of variation of ≤10% (n = 6 per day, 3 days) at both levels and calculated extraction efficiencies of 78% and 91%, respectively. The results from the samples submitted to the laboratory for BZP analysis showed 11% were positive (n = 126). The mean BZP level was 3.9 ng/mg (range, 0.4–33 ng/mg; the result was extrapolated when above the calibration). These data are the first available showing the levels expected from users of BZP.


Distribution of BZP in hair (ng/mg) from 14 positive hair samples, using LC-MS/MS detection


Hair LC-MS/MS Drugs of abuse 1-Benzylpiperazine BZP 


Benzylpiperazine (BZP) was first sold commercially as an alternative and legal drug in New Zealand around the year 2000. As it was not listed under the Misuse of Drugs Act 1975, there were no legal controls around sale or distribution. This meant that the distributors were able to market BZP as a dietary supplement without need for pre-market approval. As manufacturers and distributors also avoided claims about therapeutic effect, the tablets did not fall under the control of the Medicines Act 1981. Prior to the rise of BZP as a recreational drug, there were no licensed therapeutic uses.

In 2005, the New Zealand Government created a new classification of “Restricted Substances” with benzypiperazine (BZP) as the first example. This schedule is informally referred to as “Class D”; under this classification BZP was available to over 18-year-olds only, and there was control over the advertising and labeling of the products. Upon review of the legislation in 2007 BZP (and some associated drugs such as 3-trifluoromethylphenylpiperazine (TFMPP)) was placed into Class C1. This law came into effect on April 1 2008 and allowed a 6-month amnesty period for the possession of the drugs, after which they became illegal to possess or to sell. Class C1 is the same classification as cannabis plant material in New Zealand.

Before the reclassification, it was reported that over 20% of the New Zealand population had tried BZP at least one time [1]. With users experiencing effects similar to those experienced by users of amphetamine and “ecstasy” (methylenedioxymethamphetamine (MDMA)), piperazines like BZP and TFMPP have been classed as stimulants. Effects are said to include euphoria, an increased sense of alertness, a desire to socialize, and mild hallucinations [2, 3]. Physical side effects include a “hangover” (similar to that of alcohol), headaches, loss of appetite, stomach pains, and tremors and shakes [2]. Many users reported psychological symptoms such as trouble sleeping, loss of energy, strange thoughts, and moods swings [1, 2]. These symptoms led to over 3% of users seeking treatment following legal party pill use (via hospital emergency room, General Practitioner, or ambulance), which led to around one in 250 users being admitted to hospital. It was also reported that a number of regular users had sought treatment for dependency to the drugs (2.2% of users) [1].

The aim of this study was to produce a quantitative drug screen including BZP using a single hair extract. This was in response to an increasing demand for hair analysis on case work received by the Toxicology Group of the Institute of Environmental Science and Research (ESR), New Zealand.

This method was validated with reference to the Society of Hair Testing guidelines proposed in 2004 [4]. These guidelines suggest a recommended limit of quantitation of less than or equal to 0.2 ng/mg of hair for amphetamines. These values are interpreted by some as suggested cut-off levels below which a sample should be called negative, but, with the current technologies available, it is possible to measure accurately at levels well below this. The extraction procedure used here was previously published as part of a comparison study of instrumental techniques [5]. Another study has proposed a method for the analysis of BZP in hair using GC-MS analysis following derivatization [6]; this paper did not report the results of any users, and no such data could be found at the time of writing.

Materials and method

Reagents and materials

BZP was purchased from SLN Pharmachem, India, and BZP-D7 from BDG Synthesis, New Zealand. Solid-phase extraction cartridges (Bond Elute Certify, 130 mg sorbent mass, 120 μm particle size) were from DKSH, New Zealand. All solvents were Mallinckrodt reagent grade from Biolab, NZ.

Specimen collection

At ESR, we have been offering hair testing as a commercial assay for several years. Hair samples are sent to ESR for analysis for a variety of cases. The biggest user of the service is the New Zealand family court, for parents to show fitness to look after, or have access to, their children.

The samples sent to ESR are collected remotely by either a family doctor or a practice nurse. It is recommended that the sample is taken from the posterior vertex region of the head [4]. Following cutting, a cotton thread is tied around the root end of the hair sample, and the sample is rolled in cling wrap to ensure safe transport to the laboratory. Blank hair samples were collected from laboratory personnel.

Hair preparation

Hair samples were segmented depending on the case and the needs of the client. Generally, multiples of 2-cm segments for 6 or 12 months of drug use history were used. Each hair segment is then cut into small 1-mm pieces and approximately 20 mg accurately weighed for analysis.



The hair samples were washed prior to analysis, to remove any external contamination. Each segment was washed with methanol three times. Methanol was added (1 mL), the sample sonicated for 30 s, left to stand for 5 min, and then centrifuged for 5 min at 2,700 rpm. The solvent was removed with a clean glass Pasteur pipette without taking up the finely cut hair. Internal standard (as below) was added to each wash; this was then dried and reconstituted in mobile phase for analysis.

Calibration curve preparation

A stock mix, which included BZP, was prepared in methanol and diluted as appropriate to produce a six-point calibration curve. Concentrations used to produce the curve were 0, 0.086, 0.216, 0.43, 2.16, and 8.63 ng/mg BZP (spiked to a 20-mg blank hair sample). A blank hair sample without internal standard was prepared with each batch.

A stock solution of deuterated internal standard (BZP-D7) was made in methanol and 100 μL added to each sample (equivalent to 2.5 ng/mg).

Extraction from hair

The hair extract was prepared as previously published [5]. Briefly, acid (HCl 0.1 M, 1 mL) was added to the samples; the tube was capped tightly, vortexed briefly, and then left to stand overnight (approximately 16 h) at 37 °C. The samples were then centrifuged (2,700 rpm, 5 min) and adjusted to approximately pH 7 using NaOH (100 μL, 1 M). Phosphate buffer was added (1 mL, 0.1 M, pH 7); the tubes vortexed briefly and centrifuged (2,700 rpm, 5 min). The liquid sample was then transferred to a clean, labeled tube.

Solid-phase extraction

The solid-phase extraction (SPE) column (Bond Elut Certify) was conditioned by sequential addition of methanol (2 mL) and phosphate buffer (2 mL, 0.1 M, pH 7). The extracted sample was transferred to the SPE column and washed with water (2 mL), acetic acid (0.1 M, 1 mL), and methanol (2 mL). The column was dried for 5 min and the analytes eluted with dichloromethane: propan-2-ol and ammonium hydroxide (2 × 2 mL, 80:20:3 v/v). Acidic methanol (50 of 15 μL concentrated HCl in 25 mL methanol) was added to the collected fraction before evaporation under a gentle stream of nitrogen at room temperature.

The samples were reconstituted in 100 μL of the mobile phase (see Instrumental Analysis below) for analysis by LC-MS/MS.

Instrumental analysis

Analysis was performed using an Ionics EP10+ HSID API 365 mass spectrometer with a Dionex UltiMate 3000 HPLC system. A 10-μL aliquot of the extract was injected onto a Phenomenx Luna® SCX column (150 × 2.0 mm) with a SCX 4 × 2.0 mm guard column in place. The analysis was performed using isocratic elution, with acetonitrile (75%) and ammonium formate buffer (100 mM) + 0.5% formic acid (25%) as mobile phase at a flow rate of 200 μL/min.

The mass spectrometric detection was carried out by a using a triple quadrupole mass spectrometer equipped with a turbo ion spray atmospheric pressure interface operating in positive ionization mode, using multiple reaction monitoring mode acquisition for analyte confirmation. The transitions monitored were determined by infusion of each compound directly into the mass spectrometer. Three transitions are monitored for BZP (177.1/91.1; 177.1/85.2; 177.1/65.1) and one for the deuterated internal standard BZP-D7 (184.2/98.2).

Method performance characteristics

Within-day precision and accuracies (n = 6) and also inter-day data (n = 18) were established using blank hair samples spiked with BZP at 0.216 and 2.16 ng/mg.

Extraction efficiency (EE) was established by comparing the analyte peak areas of six extracted samples with the analyte peak areas of six unextracted samples, spiked with the same levels of BZP as above.


Method performance characteristics

The calibration curves showed linear regression coefficient of R2 > 0.99 over the range analyzed. Results for the validation are shown in Table 1. The EE was calculated as 78% and 91% at 0.216 and 2.16 ng/mg, respectively.
Table 1

BZP inter- and intra-day assay precision and accuracy values from validation experiments

Intra-day (n = 6)

Inter-day (n = 18, 3 × 6)

Mean (SD)

CV (%)

Accuracy (%)

Mean (SD)

CV (%)

Accuracy (%)

0.22 (0.02)



0.21 (0.02)



2.29 (0.09)



2.23 (0.10)



The limit of detection was not established. At the lowest calibration point, the signal-to-noise was much greater than 5:1, and our lowest calibrant (0.086 ng/mg) was approximately half the value of the maximum recommended limit of quantitation from the Society of Hair Testing for amphetamine-type stimulants (0.2 ng/mg). Thus, the method was deemed to be sufficient for its intended purpose of detecting the presence of BZP. We chose to use the lowest calibrant as our cut-off, meaning results below the calibration curve are recorded as being not detected. An example chromatogram of a blank hair matrix (top), the lowest standard (middle), and a positive sample (bottom) is shown in Fig. 1.
Fig. 1

Extracted ion chromatograms for BZP (177.1/91.1, left) and BZP-D7 (184.2/98.2, right) from analysis of a blank hair matrix (top), the low calibrant (0.086 ng/mg, middle), and a positive sample (bottom)

Hair sample results

One hundred twenty-six hair samples were analyzed, 14 (11.1%) of which were found to be positive for BZP. The range of BZP detected in these positives samples was from 0.13 to 33.3 ng/mg with the mean and median 3.88 and 0.83 ng/mg, respectively. Only one sample had a value (33.3 ng/mg) above the highest calibrant (8.63 ng/mg). The spread of data can be seen in Fig. 2.
Fig. 2

Distribution of BZP concentrations in hair (nanograms per milligram) from 14 positive samples

All of the washes were analyzed, and none of the samples had a wash-to-hair ratio of greater than 1:10, above which the result may be in part due to external contamination [7].


This is the first study to describe the analysis of BZP in hair. The extraction method is efficient and can be used for a broad spectrum analysis for drugs of abuse and prescription drugs [5]. The detection of BZP in hair samples from New Zealanders is not a surprise, given the legal availability prior to reclassification. Prior to reclassification BZP accounted for around 30% of the positive amphetamine type stimulant results in urinary drug screening at ESR [8].

The lowest concentration of calibrant in this study was 0.085 ng/mg. At this concentration, BZP had a signal-to-noise ratio on the LC-MS/MS of much greater than 5:1. At ESR, the hair case work is generally reported as qualitative, as there is not an accepted method of calculating the amount of drug used from the level detected in the hair. However, the data produced is quantitative, as shown by this paper. There have been no studies published to determine whether there may be a correlation between the amount of BZP taken and the level detected in the hair. The quantitative results may be used to determine from segmental analysis if there is an increasing or decreasing pattern of drug use with time in an individual.

Over the same time period, the other common drugs detected in our testing were methamphetamine, amphetamine, codeine, and MDMA (26%, 13%, 13%, and 6%, respectively, unpublished data). The mean level of BZP detected in these hair samples (3.88 ng/mg) is around half the level of methamphetamine from our cohort (6.6 ng/mg; range, 0.11–92 ng/mg). The methamphetamine range detected is very similar to that detected in one prior study (0.1–128 ng/mg, n = 97 [9]), while, in another large study, the range was seen to be larger and the mean higher (0.5–608.9 ng/mg, mean 21.0 ng/mg, median 10.1 ng/mg [10]), this study had a cut-off of 0.5 ng/mg, so would not include any values in the 0.1 to 0.5 ng/mg range, thus inflating the mean. No prior BZP results are published for comparison.


This paper presents the first validated assay for the analysis of BZP in hair samples using LC-MS/MS. The assay has proved to be robust with over 100 cases analyzed. The assay extraction is the base for a broad screen analysis. The use of LC-MS/MS for analysis minimizes the potential losses of the analyte by removing the derivatization step required for GC-MS analysis.



The authors thank the analytical team in the Specialized Analytical Services group of ESR.


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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Kenepuru Science CentreInstitute of Environmental Science and Research LtdPoriruaNew Zealand

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