4.1.1.  Protective measures

Avoid breathing of dust, vapours, mist or gas. Wear full-face particulate filtering respirator type N100 (US) or type P3 (EN 143) respirator cartridges when working with the solid material. Wear protective gloves, goggles and clothing. Take special care to avoid skin exposure if handling solutions and work in adequately ventilated areas. Wash hands thoroughly after handling.

4.1.2.  Emergency procedures

General advice

Immediately call a POISON CENTER or doctor/physician. Show this safety data sheet to the doctor in attendance. Move out of dangerous area.

If inhaled

Move person into fresh air. If not breathing give artificial respiration. Consult a physician.

In case of skin contact

Wash off with soap and plenty of water. Consult a physician.

In case of eye contact

Rinse thoroughly with plenty of water for at least 15 minutes and consult a physician.

If swallowed

Immediately call a POISON CENTRE or doctor/physician. Never give anything by mouth to an unconscious person. Rinse mouth with water.

4.1.3.  Spillage / Projections

Contain spillage and then collect by wet-brushing and place in container for disposal. Keep in suitable, closed containers for disposal according to local regulations.

4.4.1.  Materials and methods

Chemicals:

• Zearalenone (ZEN); BIPM Reference OGO.178a
  Supplier: First Standard, Product No. 1ST7204, Lot ALT601341

NMR Solvents:

• Dimethylsulfoxide-$d_6$ (DMSO-$d_6$); BIPM Reference OGS.027c

• Deuterated chloroform (CDCl₃); BIPM Reference OGS.026b

• Acetone-$d_6$; BIPM Reference OGS.029

Solvents were purchased from a commercial supplier and used without further treatment.

4.4.2.  Sample preparation

For qualitative NMR analyses an individual sample size of approximately $10\mathrm{mg}$ of ZEN was made up in $1\mathrm{mL}$ of deuterated solvent in a glass vial. The sample solution was mixed in a vortex shaker and transferred into NMR tubes (HG-Type: high grade class, $8\mathrm{in}$, $5\mathrm{mm}$ o.d., with PE caps) using disposable glass pasteur pipettes.

4.4.3.  NMR acquisition parameters

A JEOL ECS-400 spectrometer operating at $9.4\mathrm{T}$ ($400\mathrm{MHz}$ for proton) equipped with a direct type automatic tuning (Royal) probe was used for all data acquisition. For qualitative analyses, ¹H spectra were acquired for both solvent blank and the ZEN sample using a simple pulse-acquire sequence with the parameters presented in Table 1.

Table 1.  Acquisition parameters for exploratory ¹H analyses.

¹³C experiments were conducted using an ordinary power gated sequence (pulse-acquire in ¹³C channel with proton decoupling both during acquisition and the relaxation delay) using the parameters shown in Table 2.

Table 2.  Acquisition parameters used for ¹³C analyses.

4.4.4.  1D ¹H and ¹³C spectra

The simple ¹H and ¹³C NMR spectra of the ZEN material are shown in Figure 3 and Figure 4. The results obtained were consistent with literature assignments. [10], [11] Figure 5 shows the attached proton test (APT) ¹³C spectrum of ZEN. Inverted signals correspond to methylene or quaternary carbons and normal signals to methine or methyl carbons.

Figure 3 — ¹H NMR spectrum of the ZEN in CDCl₃.

Figure 4 — ¹³C spectrum of ZEN in CDCl₃.

Figure 5 — APT spectrum of ZEN. Down = CH₂/C_q; Up = CH/CH₃.

4.4.5.  2D NMR spectra

To confirm the identification and assignment of the signals, two-dimensional homonuclear correlated spectroscopy (COSY), heteronuclear single-quantum correlation spectroscopy (HSQC) and total correlation spectroscopy (TOCSY) spectra [11], [12] were acquired. The individual spectra are reproduced in Appendix A1.2. From the combined data the peak assignments are summarized in Table 3 below. The results are fully consistent with the literature assignments and established the identity of the main component in the material as ZEN.

Table 3.  ¹H and ¹³C peak assignments for ZEN in OGO.178.a.

4.4.6.  Residual solvent content by NMR

In the ¹H NMR spectrum of the BIPM material it was possible to detect impurity peaks not present in the solvent blank originating from residual solvents: a singlet at $5.3\mathrm{ppm}$ from dichloromethane, a singlet at $3.33\mathrm{ppm}$ from methanol and a quartet at $3.5\mathrm{ppm}$ which could be either ethanol or diethyl ether. The latter is the more likely according to previously reported chemical shifts. [13]

To obtain an accurate quantification of these small signals a spectrum was acquired using 512 transients, a relaxation delay of $60\mathrm{s}$ between scans and applying the parameters optimized for quantitative analysis of ZEN (see Table 5 in Section 5.2). From this spectrum, the mass fractions of the residual solvents were calculated from the ratio of the signal integral to that of reference peaks in the ZEN spectrum. Two possible scenarios for the origin of the quartet at $3.5\mathrm{ppm}$ (either ethanol or diethyl ether) were considered. Two different ZEN peaks were used as reference values and the average values were considered as fit-for-purpose estimates of the mass fractions of the residual solvent content. To investigate the possibility that the quantification of these residual solvent peaks was influenced by contributions from ¹³C satellite peaks of adjacent ZEN peaks an acquisition using ¹³C-decoupling and otherwise the same parameters was also performed and the results compared.

The data from the two experiments (with and without ¹³C-decoupling) with calculation relative to two different ZEN peaks provided a combined result derived from the four calculated values for the levels of each residual solvent. The measurement uncertainty for this result includes contributions from the uncertainty in the molar masses of both ZEN and the solvents in addition to the pooled variation between the different measurement procedures (2 peaks, 2 acquisitions). The difference in the combined residual solvent content due uncertainty in whether ethanol or diethyl ether is present is negligible, but in order to maintain metrological traceability, an additional uncertainty component was added to take this into account. On the basis of the observed chemical shift the more likely identity is diethyl ether. If desired the identities of the residual solvents could be independently established and quantified using headspace GC-MS based technique. The results for residual solvent content assigned by the relative NMR response and their associated measurement uncertainties are summarized in Table 4. Representative spectra showing each solvent signal relative to the adjacent ZEN peaks are given in Appendix A1.4.

Table 4.  Estimated mass fraction content of the residual solvents detected in the OGO.178a material.

5.2.1.  Materials

Chemicals

• Zearalenone (ZEN); BIPM Reference OGO.178a
  Supplier: First Standard, Product No. 1ST7204, Lot ALT601341

• Zearalanone (ZAN); BIPM Reference OGO.182a
  Supplier: First Standard, Product No. 1ST7203, Lot LZ106742

• Dimethylterephthalate (DMTP); BIPM Reference OGE.022b was used as the qNMR internal standard [16]. The mass fraction content of DMTP in the material was assigned as $999.3\pm 0.8\mathrm{mg}/\mathrm{g}\left(k=2\right)$ by qNMR at the BIPM.

NMR Solvents:

• Acetone-$d_6$; BIPM Reference OGS.029

• Dimethylsulfoxide-$d_6$ (DMSO-$d_6$); BIPM Reference OGS.027c

• Deuterated chloroform (CDCl₃); BIPM Reference OGS.026b

Deuterated solvents were purchased from a commercial supplier and used without further treatment. NMR tubes were HG-Type: high grade class, $8\mathrm{in}$, $5\mathrm{mm}$ diameter rated for use with $600\mathrm{MHz}$ spectrometers fitted with PE caps.

5.2.2.  Sample preparation

Gravimetric operations were performed using a Mettler Toledo XP2U ultramicrobalance. Prior to all weighing operations the repeatability of the balance was assessed for suitability to the preparation of qNMR samples by repeat mass determinations of an empty weigh boat. The general recommendations of Yamazaki et al [17] for qNMR sample preparation were used.

Four separate samples were prepared. The individual sample sizes were in the range $4\mathrm{mg}$ — $10\mathrm{mg}$ for the ZEN material and $2\mathrm{mg}$ to $4.5\mathrm{mg}$ for the internal standard DMTP. Each sample was separately weighed into an aluminium weighing boat and then to avoid contact of the solvent with the metal boat, the contents of both were carefully transferred into a common glass vial and each emptied boat was reweighed. The amount of ZEN and DMTP transferred into the glass vial was determined by difference and this value was used for subsequent qNMR calculations. $1\mathrm{mL}$ of deuterated solvent was added to the vial and the sample solution was mixed in a vortex shaker and checked visually for completeness of dissolution. Approximately $800\mathrm{\mu L}$ of this solution was transferred into an NMR tube (HG-Type: high grade class, $8\mathrm{in}$, $5\mathrm{mm}$ o.d., with PE cap) using a glass pasteur pipette.

5.2.3.  Choice of solvent and quantification signals

Because of the complexity of the ZEN proton spectrum in the upfield section of the spectrum ($\delta <5\mathrm{ppm}$) the potential quantification peaks are limited to those occurring at chemical shift between $5\mathrm{ppm}$ and $7\mathrm{ppm}$, corresponding to the aromatic (H-13 and H-15), olefinic (H-11 and H-12) and lactone bridge (H-3) protons.

CDCl₃, DMSO-$d_6$ and acetone-$d_6$ were investigated as possible solvents. The hydrogen peak from the phenol at position 14 overlays the signal for H-11 at $5.7\mathrm{ppm}$, rendering this peak unsuitable for quantification. The signals due to the two aromatic hydrogens centered at $6.4\mathrm{ppm}$ were associated in this material with small impurities at the baseline of the peak which were considered too close to be subtracted. The most attractive signals for quantification purposes were that at $7.0\mathrm{ppm}$ corresponding to the H-12 proton and that at $5.0\mathrm{ppm}$ due to H-3. The peak at $5.0\mathrm{ppm}$ is a complex multiplet with lower intensity compared to the peak at $7.0\mathrm{ppm}$ resulting in a lower relative signal to noise ratio. In addition all impurities in the material from either the ZEN or ZAN family will have a signal at a similar chemical shift. It was known from the LC characterization of the material (see Section 4.3) that ZAN was one of the major impurities in the material.

The peak at $7.0\mathrm{ppm}$ was judged as more suitable for quantification as it would not include any contribution from the ZAN impurity or ZAN-related impurities. However in CDCl₃ this peak is overlaid by the residual chloroform ¹³C satellite. In DMSO-$d_6$ the chemical shift of the peak moves to $6.6\mathrm{ppm}$. It is now in too close proximity to the signals for the aromatic hydrogen to be used for qNMR. However for acetone-$d_6$ the H-12 signal chemical shift remains at $7.0\mathrm{ppm}$ and the residual solvent peak is well separated from the quantification region.

An unanticipated advantage was also discovered in the use of acetone-$d_6$ as solvent. It was observed that a significant curve occurred in the baseline of the spectra of ZEN in solution in CDCl₃ or DMSO-$d_6$ which was not in evidence with spectra in acetone-$d_6$. This may simply result from a contribution to the baseline from a broad acidic hydrogen signal in the aprotic solvents that is exchanged out in solution in acetone-$d_6$ due to the unavoidable presence therein of a small amount of water associated with the solvent. Whatever the source of the interference a bias to lower values was observed when qNMR was carried out on ZEN in solution CDCl₃ and DMSO-$d_6$ compared with the value obtained in solution in acetone-$d_6$. It is strongly advised to ONLY use acetone-$d_6$ for qNMR studies of ZEN materials.

Spectra illustrating the contrast between the baseline of the NMR spectrum of ZEN in acetone-$d_6$ and CDCl₃ are reproduced in Appendix A1.6.

DMTP was selected as the internal standard selected for the qNMR study. [16] This material is readily soluble and stable in both non-polar and semi-polar solvents such as acetone-$d_6$. The signal due to the four equivalent aromatic protons in DMTP which occur as a sharp singlet at $8.0\mathrm{ppm}$ was used for quantification. The integration ratio was calculated against both the multiplet ZEN H-12 signal at $7.0\mathrm{ppm}$ and the H-3 signal at $5.0\mathrm{ppm}$. The initial qNMR result for the quantification against the signal at $5.0\mathrm{ppm}$ must be corrected for contributions from all three impurities identified by LC-methods (see Section 4.3) whereas the result using the signal at $7.0\mathrm{ppm}$ need only be corrected for contributions from 6-dehydro ZEN and cis-ZEN impurities.

5.2.4.  NMR acquisition parameters

A JEOL ECS-400 spectrometer operating at $9.4\mathrm{T}$ ($400\mathrm{MHz}$ for proton) equipped with a direct type automatic tuning (Royal) probe operating using the Delta software was used for all NMR data acquisition.

The general recommendations for optimizing spectrometer performance, determining the relevant NMR experiment parameters and undertaking a qNMR experiment as described in the BIPM Internal Standard Reference Data report for the use of DMTP for qNMR measurements [16] were followed. The final qNMR acquisition parameters are summarized in Table 5.

Table 5.  Acquisition parameters for qNMR.

5.2.5.  qNMR signal integration

A baseline correction window of eighty times the FWHM was applied to each integrated signal. The integration range start and end points were placed fifty $\mathrm{Hz}$ beyond the visible edge of each signal. Results from four independent sample mixtures each measured four times were obtained.

5.2.6.  Value assignment and measurement uncertainty

Results from four independent sample mixtures each measured four times on the day of preparation were obtained with quantification using the one proton signal due for H-12 in ZEN at $\delta =7.0$. The measurement uncertainty budget is reproduced below in Table 6. The integral ratio is the overall mean of the four replicate values obtained for each of the four samples, normalized for the different sample sizes used in their preparation. The standard uncertainty of the normalized ratio is the standard deviation of the mean based on the use of four independent samples. The relative contribution of each component to the uncertainty of the result for this material is displayed in Figure 6. The mass fraction content of “ZEN” in the material from this analysis, quantified against the $7.0\mathrm{ppm}$ NMR peak in “ZEN”, was $998.0\pm 1.8\mathrm{mg}{\mathrm{g}}^{-1}$, bearing in mind that this estimate includes the contributions from the 6-dehydro ZEN and cis-ZEN impurities.

Table 6.  Uncertainty budget for ZEN purity¹ by qNMR using DMTP in acetone-$d_6$.

Figure 6 — Relative uncertainty components: ZEN assignment using DMTP in acetone-$d_6$.

Note in the uncertainty budget that the contribution from the gravimetric operations and the purity of the internal standard are as important to the overall uncertainty of the purity assignment as the contribution due to the repeatability of the integral ratio determination.

The qNMR assignment was repeated using the same set of NMR data obtained on the day of preparation of the sample but with quantification against the one proton signal for H-3 in ZEN at $\delta 5.0$. It was also repeated using the same samples and NMR acquisition and processing parameters three and seven days after the original sample preparation in order to evaluate the stability of the ZEN in solution. The qNMR assignments were obtained for each data set with quantification against both the $\delta 7.0\mathrm{ppm}$ and $\delta 5.0\mathrm{ppm}$ signals. The combined assignments are summarized in Table 7

Table 7

5.3.1.  Materials

Chemicals:

• Zearalenone (ZEN); BIPM Reference OGO.178a
  Supplier: First Standard, Product No. 1ST7204, Lot ALT601341

• Zearalanone (ZAN); BIPM Reference OGO.182a
  Supplier: First Standard, Product No. 1ST7203, Lot LZ106742

• $\mathrm{\alpha }$-Zearalenol ($\mathrm{\alpha }$-ZEL), $\mathrm{\beta }$-Zearalenol ($\mathrm{\beta }$-ZEL), $\mathrm{\alpha }$-Zearalanol ($\mathrm{\alpha }$-ZAL), $\mathrm{\beta }$-Zearalanol ($\mathrm{\beta }$-ZAL) were all provided as $100\mathrm{ppm}$ solutions in acetonitrile by First Standard

5.3.2.  Standard solutions

Two standard solutions of mass fractions of approximately $200\mathrm{mg}/\mathrm{kg}$ were prepared using the ZEN (OGO.178) and ZAN (OGO.182) materials. Each stock solution consisted of approximately $4.2\mathrm{mg}$ of material weighed accurately and made up to the mark in a $25\mathrm{mL}$ flask with acetonitrile to a final weight of approximately $19.4\mathrm{g}$. Gravimetric operations were undertaken using a Mettler AX-504 balance. The ambient temperature during gravimetric operations was $22\mathrm{°C}$ and relative humidity 54 %. For further calculations the mass fraction content of the ZEN and ZAN was based on the purity assigned for these materials at the BIPM by qNMR. For the solutions of $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL the values provided by the supplier were used.

From these six standard solutions a high concentration mixed working solution (HMC) of ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL was gravimetrically prepared by weighing accurately approximately $72\mathrm{mg}$ of three stock solutions (ZEN, ZAN, $\mathrm{\beta }$-ZAL) and $110\mathrm{mg}$ of the other stock solutions ($\mathrm{\alpha }$-ZEL, $\mathrm{\beta }$-ZEL, $\mathrm{\alpha }$-ZAL) in a $1.5\mathrm{mL}$ HPLC vial and adding acetonitrile to a final weight of approximately $0.9\mathrm{g}$ to obtain a mixed working solution. From this high concentration mixed working solution, a middle concentration mixed working solution (MMC) was prepared by accurately weighing approximately $460\mathrm{mg}$ of the HMC solution in a $1.5\mathrm{mL}$ HPLC vial and adding water to a final weight of approximately $1\mathrm{g}$

A series of five standard dilutions for each of “day 1” and “day 2” measurements were gravimetrically prepared by further dilution of the MMC solution.

5.3.3.  LC-DAD method development

A Phenomenex Kinetex EVO C-18 100Å column with dimensions $250\times 4.6\mathrm{mm}$ and $2.6\mathrm{\mu m}$ particle size was used to separate the ZEN, ZAN and related structure compounds in the ZEN material. According to previous research [10] ZEN is of limited stability in solution in methanol and this solvent should be avoided for use in the mobile phase for chromatographic analysis of ZEN. A method using as mobile phase an acetonitrile/water gradient was optimized to achieve adequate resolution of the six components of the test mixture in the smallest possible runtime.

A chromatogram of the separation of the ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL HMC standard solution obtained using this method with detection at $274\mathrm{nm}$ is shown in Figure 7.

Figure 7 — Chromatographic separation of the test mixture with LC-DAD detection

5.3.4.  LC-DAD method performance

Method validation consisted of the evaluation of the method limit of detection (LOD), limit of quantification (LOQ), linearity, sensitivity, repeatability and intermediate precision.

Performance characteristics (limits of detection (LOD) (S/N = 3), limits of quantification (LOQ) (S/N = 6), sensitivity, etc. were calculated corresponding to the 95 % — confidence level (CI) of the y-intercept according to the German standard DIN 32645 for each of ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL. The characteristics were evaluated from the calibration curves obtained from triplicate determinations of standard solutions.

In total, five mass fraction levels were analysed based on the amount of impurity estimated to be present in the ZEN material. The results for ZEN and ZAN are displayed in Table 8.

Table 8.  Performance characteristics for ZEN and ZAN. Values in brackets and “CI (95 %)” values were obtained from calculations using the tool “Validata”.

5.3.5.  Impurities in ZEN by LC-DAD

Two samples of the ZEN source material OGO.178a were analysed for related impurities using this method. About $4.2\mathrm{mg}$ of the ZEN material was transferred by aluminium weighing boat into a tared class A $25\mathrm{mL}$ volumetric, taken up in pure acetonitrile and allowed to stand over night at $4\mathrm{°C}$. Aliquots of each solution were transferred into HPLC screw top vials for analysis. The target mass fraction content of ZEN in the solution was aimed to be about $200\mathrm{\mu g}{\mathrm{g}}^{-1}$ in order to be able to detect low levels of related structure impurities. The mass fraction of each identified impurity was derived by external calibration over an appropriate linear mass fraction range.

For a ZEN sample at a mass fraction of $200\mathrm{\mu g}{\mathrm{g}}^{-1}$ for the major component, 0.1 % (or $0.2\mathrm{\mu g}{\mathrm{g}}^{-1}$) of an impurity corresponds to an absolute mass fraction of $200\mathrm{ng}{\mathrm{g}}^{-1}$. The absolute LODs corresponding to 3*S/N approach for the absorbance at $274\mathrm{nm}$ range from $53\mathrm{ng}{\mathrm{g}}^{-1}$ for $\mathrm{\alpha }$-ZEL to $127\mathrm{ng}{\mathrm{g}}^{-1}$ for ZAN, giving confidence that this method would be able to detect resolved ZEN-related impurities present at relative levels above 0.1 %.

The chromatogram obtained for the ZEN material in solution in acetonitrile at $200\mathrm{\mu g}{\mathrm{g}}^{-1}$ is shown in Figure 8. The upper pane shows the $274\mathrm{nm}$ response and the lower pane the TIC obtained by a parallel LC-MS/MS analysis of the same solution. Two impurities were detected using the LC-DAD method. The impurity eluting at $28.5\min$ was identified as ZAN based on its retention time and this assignment was subsequently confirmed by LC-MS/MS. The other impurity corresponded closely but not exactly to the retention time $\mathrm{\alpha }$-ZEL. Subsequent LC-MS/MS analysis identified this impurity as 6-dehydroZEN.

Figure 8 — Chromatogram of LC-DAD (top) and LC-MS/MS (bottom) analysis of a solution of the ZEN OGO.178a source material

No significant amounts of $\mathrm{\alpha }/\mathrm{\beta }$-ZAL or $\mathrm{\alpha }/\mathrm{\beta }$-ZEL were observed in the ZEN material by LC-DAD and this conclusion was subsequently confirmed by LC-MS/MS analysis.

The quantification of the ZAN content in the ZEN material was undertaken using an external calibration curve based on the available ZAN standard. For the 6-dehydro ZEN a calibration curve based on $\mathrm{\alpha }$-ZEL was used since they had similar retention time and no authentic standard was available for the impurity. The impurity mass fraction values and expanded measurement uncertainties as determined by the LC-DAD method are shown in Table 9.

Table 9.  Related structure impurity content of OGO.178a by LC-DAD

5.3.6.  LC-MS/MS method development

A method of liquid chromatography with tandem mass spectrometry (LC-MS/MS) was also developed for the simultaneous determination of ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL in the ZEN material. The same column and gradient method described for the LC-DAD analysis were used as the DAD and MS/MS measurements were undertaken in tandem on column eluant from the same LC system.

The MS parameters in negative electrospray ionization (ESI neg.) mode were optimised by direct infusion into the ionization source of the QTrap of single LC standards of ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL and the MRM parameters of each compound were optimized. Each measurement using the optimized parameters was undertaken in triplicate. Optimized intensity was obtained at capillary voltage of $-\mathrm{4 500}\mathrm{V}$ and source temperature of $550\mathrm{°C}$. Nitrogen was used as the ion source gas, curtain gas and collision gas. The Gas 1 and Gas 2 of ion source were at 55 psi and 50 psi; the curtain gas (CUR) was set at 15 psi. The Collision Gas (CAD) was set at Mid. Table 10 lists the results for each compound’s optimization in MRM mode.

The transitions of impurities in the material for which no authentic sample was available (6-dehydroZEN, dihydroxy-6-dehydronsZEN, cis-ZEN) were determined by direct observation.

Table 10.  Ions transitions and MS/MS parameters of ZEN and its impurities in MRM mode

The TIC chromatogram of the ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL standard solution using this method is shown in Figure 9.

Figure 9 — TIC chromatogram of the standard mixture.

5.3.7.  LC-MS/MS method performance

Method validation consisted of the evaluation of the limit of detection (LOD), limit of quantification (LOQ), linearity, sensitivity, repeatability and intermediate precision.

Performance characteristics (limits of detection (LOD) (S/N = 3), limits of quantification (LOQ) (S/N = 6), sensitivity, etc. of the method were calculated corresponding to the 95 % — confidence level (CI) of the y-intercept according to the German standard DIN 32645 for ZEN, ZAN, $\mathrm{\alpha }/\mathrm{\beta }$-ZAL and $\mathrm{\alpha }/\mathrm{\beta }$-ZEL. The characteristics were evaluated from the calibration curves obtained from triplicate determinations of standard solutions.

In total, five mass fraction levels were analysed based on the amount of impurity estimated to be potentially present in the ZEN material. The method performance for ZEN and ZAN is displayed in Table 11.

Table 11.  Performance characteristics for ZEN and ZAN. Values in brackets and “CI (95%)” values were obtained from calculations using the tool “Validata”

The linearity of the working range was evaluated by constructing calibration curves at five different concentration levels. In order not to reject the linearity hypothesis, a determination coefficient (r²) of at least 0.999 was required. Sensitivity was also determined from the calibration curve, being equal to the angular coefficient.

Note that the LOD/LOQ estimates for the LC-MS/MS method shown in Table 11 are an order of magnitude smaller than those for the LC-DAD analysis of the same samples listed in Table 10.

5.3.8.  Impurities in ZEN by LC-MS/MS

The MS3-IDA-EPI mode of the Qtrap4000 was applied to identify these impurities with a Valco valve function included in the method to cut off the main component from introduction into the ionization chamber.

The HPLC chromatography conditions were listed in the following:

Column

Kinetex C18 ($2.6\mathrm{\mu m}\cdot 150\mathrm{mm}\cdot 3.0\mathrm{mm}$) OGLC056

Column temperature

$35\mathrm{°C}$

Mobile phase

Acetonitrile+MeOH+H₂O (with 10 mM NH₄AC) =10+45+45

Operation mode

isocratic

Flow rate

$0.25\mathrm{mL}/\min$

Injection Mode

Standard

Injection volume

$2.0\mathrm{\mu L}$

Duration

$30.0\min$

A solution of approximately $1.0\mathrm{mg}{\mathrm{g}}^{-1}$ of ZEN was prepared by accurately weighing approximately $7.7\mathrm{mg}$ of material into a $10\mathrm{mL}$ flask and adding methanol to a final weight of approximately $7.85\mathrm{g}$. The calculated mass fraction of the ZEN material in the solution was $977.9\mathrm{\mu g}{\mathrm{g}}^{-1}$. To avoid contamination of the LC-MS/MS instrument by the high content of ZEN component, the LC method was slightly changed. After chromatographic separation the mobile phase was switched to waste during elution of the ZEN component, to cut out this peak from the ionization chamber so as to be able to better detect and analyse the minor impurities present in the material. Figure 10 shows the TICs of the ZEN solution in the MS3-IDA-EPI mode of the Qtrap4000 in the upper pane with the LC-DAD response at $274\mathrm{nm}$ in the lower pane. Four impurities, marked as impurity 1, 2, 3 and 4, were observed in the TIC. Two of these corresponded to peaks also seen at $274\mathrm{nm}$ using the LC-DAD. After comparison with the authentic standard, the impurity 3 was confirmed as ZAN. Impurity 4 has the same precursor ion and fragment ions as ZEN and was identified as cis-ZEN. The precursor ions and fragmental ions of the main component (ZEN) and the four impurities are listed in Table 12.

Figure 10 — TIC chromatogram (upper pane) and LC-DAD (lower pane) of OGO.178.a

Table 12.  Identification of impurities in ZEN material with QTRAP-MS

Quantitative estimates of the three major impurities present in the ZEN material identified by LC-MS/MS were undertaken. The quantification of the ZAN content was undertaken using an external calibration curve prepared with the ZAN standard. As no authentic standards were available for the other impurities the quantification of 6-DehydroZEN was undertaken using the $\mathrm{\alpha }$-ZEL calibration curve and for cis- ZEN using the ZEN calibration curve. The impurity mass fraction values and corresponding expanded measurement uncertainties are summarised in Table 13.

Table 13.  Related structure impurity content of ZEN material by LC-MS/MS