Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 381–386

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Determination of the purity of pharmaceutical reference materials by 1 H NMR using the standardless PULCON methodology Yulia B. Monakhova a,b,c , Matthias Kohl-Himmelseher a , Thomas Kuballa a , Dirk W. Lachenmeier a,∗ a

Chemisches und Veterinäruntersuchungsamt (CVUA) Karlsruhe, Weissenburger Strasse 3, 76187 Karlsruhe, Germany Bruker Biospin GmbH, Silberstreifen, 76287 Rheinstetten, Germany c Institute of Chemistry, Saratov State University, Astrakhanskaya Street 83, 410012 Saratov, Russia b

a r t i c l e

i n f o

Article history: Received 14 July 2014 Received in revised form 14 August 2014 Accepted 18 August 2014 Available online 26 August 2014 Chemical compounds studied in this article: Ibandronic acid (PubChem CID: 60852) Amantadine (PubChem CID: 2130) Ambroxol (PubChem CID: 2132) Lercanidipine (PubChem CID: 65866) Keywords: H NMR PULCON methodology Ibandronic acid Amantadine Ambroxol 1

a b s t r a c t A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15 min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis. The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the purpose of quantitative analysis because of the direct proportionality of signal intensity to the number of resonating nuclei and has found many applications in analytical chemistry [1–5]. Usually quantification is provided using a signal from a chemical reference standard added to the sample (internal referencing). However, this compound has to fulfill several requirements, which makes it sometimes difficult to find a suitable reference compound and, therefore, requires investment in method development [6,7]. This is especially challenging in biological applications due to problems of overlapping signals and because in these cases adding substances bears the risk of chemical interaction with biological macromolecules [6].

∗ Corresponding author. Tel.: +49 721 926 5434; fax: +49 721 926 5539. E-mail address: [email protected] (D.W. Lachenmeier). http://dx.doi.org/10.1016/j.jpba.2014.08.024 0731-7085/© 2014 Elsevier B.V. All rights reserved.

An alternative to the internal chemical reference calibration has been proposed by Akoka et al. [8]. The ERETIC1 method (Electronic REference To access In vivo Concentrations) provides a reference signal, synthesized by an electronic device, which can be used for the determination of absolute concentrations [8]. The advantage over the above-mentioned internal referencing is that nothing is added to the sample and the reference signal frequency can be freely chosen within a transparent region of the spectrum (i.e. a region where there are no matrix signals). Some studies demonstrated the accuracy and precision of the ERETIC1 method for quantification of reference compounds, sugar surfactants, taurine in energy drinks, protoberberine alkaloids in traditional Chinese medicines as well as fatty acids, phosholipids and lipids in microalgae samples [4,7–11]. Some applications of the ERETIC1 methodology are known in solid state NMR [12–15] and 2D NMR experiments [16,17]. The drawbacks of the method are that the artificial signal has to be properly and regularly correlated and the method itself requires additional specialized hardware and modifications in spectrometer setup [6,8,18]. In solid state NMR,

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temperature dependence of the signal intensity sometimes additionally prevents precise concentration estimation [12]. Another promising approach used in this paper is to perform qNMR with an external standard via sequential analysis on a given instrument of a separate solution containing a compound (not necessary the target analyte) in a defined concentration [5,6]. The quantification is performed using the PULCON (PULse length based CONcentration determination) methodology (different from ERETIC1), which is based on the principle of reciprocity, meaning that the NMR signal intensity for a sample compound in a given coil is inversely proportional to 90◦ or 360◦ pulse length [6,20]. PULCON compensates signal intensities for losses in coil sensitivity due to sample-to-sample changes in dielectric properties (ionic strength) and does not require specialized equipment to be performed [19]. Consequently, the advantages of the ERETIC1 and PULCON methods over the internal referencing are no overlap, no chemical reactions and no need to develop a compatible solvent/analyte/standard combination. However, the literature on application of the PULCON method is rather scarce [5–7,20,21]. The objective of the present investigation was to apply a PULCON NMR method for precise measurements of purity of pharmaceutical reference standards and to compare this methodology with NMR quantification with internal standard and reference methods. Our aim was to evaluate the applicability of PULCON methodology in routine medicines control.

2. Experimental 2.1. Samples and sample preparation Eight samples were analyzed in our capacity as official medicines control laboratory including ibandronic acid (Sample S1, Synthon Chemicals, Wolfen, Germany), amantadine–HCl (Sample S6, Merckle, Blaubeuren, Germany), lercanidipine (Sample S7, Merckle, Germany), ambroxol–HCl (Sample S8, C.P.M., Germany). Four medicinal preparations with ibandronic acid as an active ingredient (Samples S2–S5) were provided by the regional administrative authorities in the German Federal State of Baden-Württemberg for routine drug surveillance. An overview of the analyzed samples is given in Table 1. All solvents and reagents used were in pro analysis quality. The following NMR buffers were prepared: pH 6.7 (500 mg of NaH2 PO4 , 2.10 ml KOH (1 M) and 1 mg of NaN3 in 25 ml of pure water); pH 1.9 (10 g of KH2 PO4 , 72 ml H3 PO4 (10 M) and 10 mg of NaN3 in 500 ml of pure water). The exact pH values were adjusted using H3 PO4 or NaOH. For the preparation of ibandronic acid samples (S1–S5), about 20 mg was dissolved in 1.0 ml of NMR buffer (pH 6.7). For the quantification with 3,5-dinitrobenzoic acid (purity 99%) as the internal standard, 600 ␮l of the above-mentioned solution was mixed with 100 ␮l of 3,5-dinitrobenzoic acid solution (about 5700 mg l−1 ), 200 ␮l of NMR buffer (pH 6.7) and 100 ␮l of 0.1% sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) (purity 98%) solution in water. For the amantadine–HCl (S6) and ambroxol–HCl (S7) samples, the following sample preparation was used: about 20 mg of sample was dissolved in 5.0 ml of NMR buffer (pH 1.9). Then 600 ␮l of the mixture was mixed with 100 ␮l of 3,5-dinitrobenzoic acid and 100 ␮l of TSP solutions (see above). As lercanidipin is soluble only in non-polar solvents, a stock solution was prepared by dilution of approximately 20 mg of the sample S8 in 5.0 ml CDCl3 containing 0.1% tetramethylsilan (TMS) (purity 99%). Then 500 ␮l of the mixture was mixed with 100 ␮l of 2,3,4,5-tetrachloronitrobenzene (purity 98%) solution (about 10,000 mg l−1 ).

The same sample preparation was performed for the PULCON qNMR method except the addition of internal standard (3,5dinitrobenzoic acid or 2,3,4,5-tetrachloronitrobenzene) solution. In all cases 600 ␮l of the final solution were poured into an NMR tube for direct measurement. 2.2. NMR method All 1 H NMR measurements were performed using a Bruker Avance 400 Ultrashield spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5-mm SEI probe with Z-gradient coils and a Bruker Automatic Sample Changer (B-ACS 120). All spectra were acquired at 300.0 K. NMR spectra of aqueous solutions were acquired using an optimized water suppression 1D noesygppr1d pulse sequence [2], where delay D7 (delay for inversion recovery) can be varied to achieve full relaxation of the targeted protons. During preliminary experiments, D7 was optimized via successive prolongation of the D7 time from 10 to 60 s until the integrated signal area remains constant and this optimum value was chosen for further experiments. NMR spectra of chloroform solutions were acquired using the Bruker standard zg30 pulse sequence with 64 scans and 2 prior dummy scans (DS). Other acquisition parameters were described elsewhere [22]. All spectra were recorded in the baseopt mode (generates a smooth baseline at zero without offset). The acquisition parameters (pulse program, time domain (TD), DS, acquisition time (AQ), sweep width (SW), receiver gain (RG), and size of spectrum (SI)) were kept constant for reference and sample spectra for PULCON measurements. For each sample during spectra acquisition, the 90◦ pulse width (P1 in Bruker terminology) was automatically estimated. This value was taken into account during quantitative analysis (see Section 2.3). The data were acquired automatically under the control of ICONNMR and Sample Track (Bruker Biospin, Rheinstetten, Germany), requiring 15–20 min per sample. All NMR spectra were phased, baseline-corrected and manually integrated using Topspin 3.1 (Bruker Biospin, Rheinstetten, Germany). 2.3. qNMR based on internal standard For quantification with an internal standard, the well-known equation was used, which includes molar weight, integral, number of protons of reference compound and analyte as well as purity and concentration of the reference [2,7,23]: mx =

MWx nHstd Ax · · · mstd , MWstd nHx Astd

(1)

where mx and mstd are the masses (weights in g), MWx and MWstd are the molecular weights in g/mol, nHx and nHstd are the numbers of protons generating the selected signals for integration, Ax and Astd are the areas for the selected peaks of the sample and the reference standard. The quantity of targeted substances is calculated by integrating a particular signal of interest and two signals of 3,5-dinitrobenzoic acid (at ı 9.17 ppm or ı 9.02 ppm) or the signal of 2,3,4,5tetrachloronitrobenzene (at ı 7.74 ppm). 2.4. qNMR using PULCON principle For chloroform solutions, the ERETIC factor was calculated using ethylbenzene solution in CDCl3 (4780 mg l−1 ) using the following equation: ERETIC =

I · SW · MW , SI · C · N

(2)

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Table 1 Quantification of purity of pharmacologically active compounds using different methods. Sample

S1 S2 S3 S4 S5 S6 S7 S8 a b c d

Sample description

Reference standard Medicinal productd Medicinal product Medicinal product Medicinal product Reference standard Reference standard Reference standard

Compound

Ibandronic acid Ibandronic acid Ibandronic acid Ibandronic acid Ibandronic acid Amantadine–HCl Ambroxol–HCl Lercanidipine

Specification (certified value)

–(100%) 95–105% (100%) 95–105% (100%) 95–105% (100%) 95–105% (98%) 98–101% (100.0%) 99–101% (100.6%) 99–101% (99.3%)

NMR (internal standard)

NMR (PULCON)

Purity [%]a

Purity [%]

94.7/96.2 93.8/95.6 96.0/98.7 94.7/96.0 93.6/96.9 97.1/97.9 102.0/105.1 99.5/104.6

CV [%] b 1.0 (6) 1.3 (6) 2.0 (6) 1.3 (6) 2.9 (6) 0.8 (3) 1.7 (6) 2.7 (6)

99.6/100.9 94.3/96.1 96.9/99.5 94.9/97.2 94.8/96.7 96.8/98.9 99.6/102.8 96.8/101.7

HPLC

CV [%] 0.7 (6) 1.9 (6) 1.6 (6) 2.4 (6) 2.2 (6) 2.5 (3) 1.8 (6) 2.8 (5)

Purity [%] c

– 99.3 103.0 99.9 100.7 –c 100 100

CV [%] –c 2.0 (3) 0.2 (3) 2.5 (3) 0.3 (3) –c 0.2 (2) 0.2 (3)

Average/maximum value. Number of signals used for variation coefficient calculation. Not conducted. For medicinal products (ibandronic acid solutions for injection), percentages relate to the labeled amount in the pharmaceutical preparation.

where I is the absolute integral, SW is the sweep width (20.5503), MW is the molecular weight (106.17), N is the numbers of protons generating the selected signal, C is the concentration, SI is the size of the real spectrum (32,768). The following signals of ethylbenzene were integrated: ␦ 7.24–7.11 ppm (N = 3, m), ␦ 2.75–2.54 ppm (N = 2, q), ı 1.31–1.11 ppm (N = 3, t) and the average ERETIC factor was used for further calculations. The ERETIC factor for aqueous solutions was assessed utilizing citric acid (purity 99%) solution (2045 mg l−1 in D2 O) by Eq. (2). The multiplet at ␦ 3.05–2.75 ppm (N = 4) was utilized for calculations. The analyte concentrations in the samples were calculated using the following equation: C=

I · SW · MW · NSRS · P1X , SI · ERETIC · N · NSX · DF · P1RS

(3)

where I is the absolute integral, DF is the dilution factor (if necessary), P1X and P1RS are the length of the 1 H 90◦ pulses, NSX and NSRS are the number of scans for spectrum of sample (X) and the reference (RS), SI – is the size of real spectrum (analyte spectrum), ERETIC – eretic factor (see Eq. (2)). For each sample, the average concentration along with the coefficient of variation (CV) was assessed using different NMR signals of the targeted compounds. To check the quality of the value of the ERETIC factor in terms of precise initial balance, sample preparation and NMR experiment, the concentration of compounds in control solutions (citric acid, 2045 mg l−1 , D2 O, multiplet at ı 3.05–2.75 ppm, N = 4; cyclohexane, 2000 mg l−1 , CDCl3 , singlet at ı 1.50–1.31 ppm, N = 12 and 2,3,4,5-tetrachloronitrobenzene 9100 mg l−1 , CDCl3 , singlet at ı 7.83–7.66 ppm, N = 4) was determined on the same day as the samples. The recovery had to be 100 ± 3% to perform further calculations for the pharmaceutical samples. 2.5. Reference HPLC methods Due to good solubility of ibandronic acid (sodium salt) in water, the reference substance as well as liquid samples were diluted and analyzed by HPLC using a refractive index (RI) detector. The injection volume was 100 ␮l, and the eluent consisted of the mixture of water:acetonitrile:EDTA (995:5:0.1) with an addition of 0.1% TFA (isocratic mode). The column used was Hypersil BDS 125 mm × 4 mm, 3 ␮m particle size (ThermoQuest, Egelsbach, Germany), the flow rate was 0.3 ml min−1 . The relevant retention time was about 6 min. Ambroxol (as hydrochloride) was diluted in an equal mixture of acetonitrile and phosphate buffer (pH 7.0) and was analyzed by a HPLC-DAD system (Agilent 1100) at the wavelength of 248 nm. The column used was Purospher 250 mm × 4 mm, 5 ␮m particle size

(Merck, Darmstadt, Germany), the flow rate 1.3 ml/min. Amantadin had been checked by GC according to manufacturer’s information (solution in water with ISTD 2-phenylethylamine and shaking out with toluene after alkalization) and analyzed by a GC-FID system (HP 5890), fused silica capillary column with dimethylpolysiloxane, length 30 m, i.d. 0.25 mm, He at 100 ml min−1 , temperature injector 250 ◦ C, oven 80 ◦ C. Lercanidipine (as hydrochloride) was dissolved in a mixture of acetonitrile, water and triethanolamine (mobile phase A 10: 90: 0.2 (v/v), pH 2.3 with phosphoric acid, mobile phase B 90: 10: 0.2 (v/v), pH 2,3 with phosphoric acid) and analyzed by a HPLC-DADsystem (Agilent 1100) at the wavelength of 230 nm. The column used was Zorbax SB C18 150 mm × 3 mm, 5 ␮m particle size (Agilent, Böblingen, Germany), the flow rate 0.85 ml min−1 (gradient mode with two mixtures of acetonitrile:water:triethanolamine in different composition). This method was also suitable to detect impurities and degradation products. All HPLC methods were validated and accredited under ISO/IEC 17025. 3. Results and discussion 3.1. Quantification of ibandronic acid Ibandronate is a potent, nitrogen-containing bisphosphonate with proven efficacy in the treatment of metastatic bone disease and postmenopausal osteoporosis [24,25]. Direct LC measurement of ibandronic acid with UV or fluorescence detection is impossible, because the molecule does not contain chromophores. Therefore, the LC assays rely on RI-detection [26]. Potential alternatives are GS-MS, HPLC–MS/MS and ELISA, which have been used to measure ibandronate concentrations in bones and biological fluids [27,28]. Recently capillary zone electrophoretic and ion chromatographic methods were developed for the analysis of impurities in technicalgrade ibandronate [29]. However, for the control of the purity of medicinal products and reference materials, the sensitivity reached by these methods (up to 10 pg ml−1 for ELISA [27]) is not necessary but it is more important to obtain fast and precise results with the opportunity to identify possible impurities. NMR could fulfill these requirements [1,3]. The NMR spectrum of one of five ibandronic acid samples (S1) is shown in Fig. 1a. For quantification, six observable resonances were used: at ı 3.51 ppm (N = 1, m), ı 3.02 ppm (N = 1, m), ı 2.83 (N = 3, s), ı 1.72 (N = 2, m), ı 1.35 (N = 4, m), ı 0.90 (N = 3, t). The results showed satisfactory purity values for all investigated samples of ibandronic acid (Table 1). The average purity varied between 93.6% and 96.0% for qNMR with internal standard. Using the PULCON methodology, the values were higher (and, therefore,

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800000

B

800000

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0

0 9

400000

4

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Intensity [A.U.]

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Fig. 1. NMR spectra of the investigated samples: ibandronic acid (S1, A), amantadine (S6, B), ambroxol (S7, C), lercanidipine (S8, D).

better) and were between 94.8% and 99.6% (Table 1). However, ttest did not recognize any statistical difference between the two quantitative NMR methods. The coefficients of variation (CVs) calculated using six different signals of ibandronic acid were always lower than 3% for the two NMR methods showing the high reproducibility of NMR (Table 1). Interestingly, in all cases the maximum purity values were obtained by integrating the resonance at ı 1.72 ppm and, therefore, this signal is recommended for routine quantification. PULCON maximum concentrations were in agreement with the sample specifications and were close to the reference HPLC values (Table 1). Therefore, it was shown that direct measurements of ibandronic acid concentrations by PULCON can be very precise and accurate. Furthermore, our data clearly demonstrated that citric acid is a suitable calibrant for qNMR experiments in aqueous solutions. Due to its low price, high purity, easy handling, and good solubility, citric acid was selected as the most adequate candidate for qNMR procedures. Different conductivities of the standard and samples are compensated by the changing of 90◦ pulse length and are taken into account in Eq. (3). This necessary compensation is not provided in the ERETIC1 methodology and many factors in coil loading (pH, salt content or probe tuning and matching) can influence the result [6]. 3.2. Quantification of purity of reference compounds To further check the performance of the PULCON NMR method, three commercial reference substances (ambroxol, amantadine,

lercanidipine) were analyzed, for which analytical certificates and the results of in-house reference analysis were available. All three compounds are used as medicines for different kinds of diseases. For example, amantadine is utilized as an antiviral medicine for influenza [30] and as an anti-Parkinson agent [31]. For the determination of amantadine in biological samples, chromatography (HPLC–MS and GC-FID) or spectroscopy (ion mobility spectrometry and fluorescence) with obligatory derivatization and/or extraction steps are used [32–36]. Ambroxol is a non-prescription drug in the form of tablets and tinctures for the treatment of acute and chronic bronchial diseases associated with disorders of mucous production and transport [36]. A commonly applied analytical method for the quantification of this compound is HPLC with photodiode array detector [37–40]. Finally, lercanidipine is a calcium channel blocker (dihydropyridine type) used for the treatment of diseases associated with high blood pressure [41]. Specific and sensitive LC-MS/MS, HPLC-UV and voltammetric methods are known to control the content of lercanidipine in biological fluids and pharmaceutical formulations [42,43]. It should be mentioned that qNMR has never been used for the quantification of these three compounds. The spectra of samples (S6–S8) are shown in Fig. 1b–d. The following signals of amantadine were integrated: ı 2.15 ppm (N = 3, s), ı 1.86 ppm (N = 6, s), ı 1.68 (N = 6, m) (Fig. 1b). For quantification of ambroxol six resonances were integrated: at ı 4.24 ppm (N = 2, s), ı 3.23 ppm (N = 1, m), ı 2.22 (N = 2, d), ı 2.07 (N = 2, d), ı 1.54 (N = 2, m), ı 1.37 (N = 2, m) (Fig. 1c). Finally, signals at ı 11.92 ppm (N = 1,

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d), ı 8.03 ppm (N = 2, m), ı 7.60 ppm (N = 1, m), ı 5.00 ppm (N = 1, d), ı 2.95 ppm (N = 2, d), ␦ 2.57 ppm (N = 5, m) were integrated for lercanidipine (Fig. 1d). After data acquisition and processing, the average recoveries for the PULCON method were found to be 96.8% for amantadine and lercanidipine and 99.6% for ambroxol (Table 1). Maximum values were even better: 98.9% for amantadine, 101.7% for lercanidipine and 102.8% for ambroxol. Comparable but slightly worse recoveries were obtained using internal standard (Table 1). The purity obtained by PULCON for the ambroxol sample (99.6%) was found to be very close to the targeted HPLC value (100%). As for ibandronic acid, CVs were smaller than 3% for all three examples. t-Test suggested no statistical difference between the two quantitative NMR methods. Based on the results for the ambroxol sample, it can also be postulated that the PULCON quantification is also applicable for quantification in volatile solvents such as CDCl3 , which has previously been not recommended due to potential errors caused by vaporizing effects during preparation of the stock solutions, pipetting or spectroscopic measurements [5]. The standardless 1 H NMR method using PULCON calibration was shown to be suitable for checking the specifications of commercial reference products even in the presence of bound water (monohydrate of sodium salt of ibandronic acid) or inorganic residuals (ambroxol hydrochloride and amantadine hydrochloride) provided that the composition is known and that appropriate signals without overlapping exist for integration. The clear advantage of the PULCON methodology over internal referencing is that a single calibration may be reused for multiple samples over a long period, provided system suitability is demonstrated on the day of data acquisition. For this purposes, the solutions of citric acid (in D2 O), and cyclohexane or 2,3,4,5-tetrachloronitrobenzene (in CDCl3 ) are recommended.

4. Conclusion For the majority of modern analytical methods (such as HPLC or GC/MS) reference substances are required. However, problems with obtaining and storage of standard materials may arise. For example, many substances are expensive, extremely difficult to obtain because they are no longer manufactured in a given country (Germany in our case) or are under patent protection. Moreover, many of these substances are light, air, temperature-sensitive or hygroscopic, and, therefore, cannot be stored during a long time and the purity of such substances has to be often reconfirmed. The problem is becoming more challenging for laboratories (like ours), which are responsible for controlling a broad range of authorized or registered drugs (about 10,000 compounds, legal market) or samples seized from custom control or criminal actions (illegal market, more than 42,000 compounds). NMR spectroscopy is probably a unique inherent quantitative technique, because due to the linear relationship between signal area and absolute concentration, it is suitable for (but not restricted to) quantification of reference standards and active ingredients in drugs. NMR is promising for controlling the purity of reference substances because it is specific and possible interferences and/or degradation products can be easily identified and quantified (in comparison with coelution of structurally related compounds in HPLC). Also, only a small amount of substance is required for NMR analysis, which compensates the high costs of standards. Finally, NMR is the only method to verify the identity and provide quantification of any drug without having reference material itself at the disposal. Recent advances in NMR method development showed that completely standardless quantification of reference compounds

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