Quantitative Analysis of Multicomponent Mixtures of Over-the-Counter

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Quantitative Analysis of Multicomponent Mixtures of Over-theCounter Pain Killer Drugs by Low-Field NMR Spectroscopy Aleksandra Zivkovic,*,† Jan Josef Bandolik,† Alexander Jan Skerhut,† Christina Coesfeld,† Momir Prascevic,‡ Ljiljana Zivkovic,‡ and Holger Stark*,† †

Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätstraße 1, Duesseldorf 40225, Germany ‡ Faculty of Occupational Safety, University of Nis, Carnojevica 10A, 18000 Nis, Serbia S Supporting Information *

ABSTRACT: Marketed pain relief drugs with one to three biologically active components, as well as mixtures of these ingredients, were qualitatively and quantitatively analyzed in an undergraduate student lab using a compact, low-field 1H NMR spectrometer. The students successfully analyzed more than 50 self-made sample mixtures with two or three components as well as the two marketed tablet formulations containing acetylsalicylic acid/L-ascorbic acid, or acetylsalicylic acid/ paracetamol (acetaminophen)/caffeine. The NMR-based quantification is an attractive application of the technique, as well as a helpful introduction to NMR spectroscopic applications in life sciences. Problem-based learning on NMR techniques on commonly known drugs provided students the opportunity to develop and improve their skills in solving 1H NMR problems. KEYWORDS: Second-Year Undergraduate, Analytical Chemistry, Hands-On Learning/Manipulatives, Problem Solving/Decision Making, Drugs/Pharmaceuticals, NMR Spectroscopy, Instrumental Methods, Medicinal Chemistry, Quantitative Analysis, Qualitative Analysis

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INTRODUCTION Modern analytical chemistry cannot be imagined without 1H NMR spectroscopy. Most often, it is used for the structural characterization of the compounds, but the variety of information that can be obtained from a simple 1H NMR measurement is very large. This complex information on chemical shift and integral and multiplicity of signals, which can simultaneously be obtained from a single 1H NMR measurement, is a hindrance for a number of students in accessing basic knowledge in NMR spectroscopic techniques. Integrals are one of the key functions in 1H NMR measurements to determine the number of hydrogens. The area under the curve of the signals (integrals) is directly proportional to the number of protons the signal describes. This fact represents the theoretical background of the experiment described herein.1,2 We have introduced low-field, benchtop NMR spectroscopy for students to achieve their own measurements with the apparatus. There are a few NMR benchtop spectrometers that are budget-priced, run at relatively low costs, and can be used for low-field NMR spectroscopy as teaching tool.3,4 As the importance of teaching NMR spectroscopy is very high, there is a need for developing new NMR experiments for the student lab. Some examples of such experiments can be found in the literature.5,6 Here, we use 1H NMR spectroscopy © XXXX American Chemical Society and Division of Chemical Education, Inc.

for quantitative ratio determination of the over-the-counter (OTC) components without the use of internal standards. There are a few experiments that were developed for the student lab, but mostly they use simple solvent mixtures (as, for example, ethyl acetate, acetone, isopropanol, etc.) and internal standards in the determination. The internal standards used most commonly are dichloromethane, trichloroethane, tetrachlorosilane, and so on.5 The main principle of the experiment relies on the proportionality of the integrals and number of protons, as for previously described experiments as well. The main goal of our experiment is for students to determine the mass ratio of the components in the two- and three-component mixtures. Students determine OTC drugs not only in the mixtures prepared for them, but also in their marketed tablet formulations available in the pharmacy. Students are shown that, in addition to simple solvent mixtures, the quality and quantity of more complex drug structures can also be determined. Here, we would stress that our main pedagogic goal is to introduce the 1H NMR technique to students and deepen their understanding for both integration and chemical Received: February 10, 2016 Revised: November 8, 2016

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DOI: 10.1021/acs.jchemed.6b00105 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Communication

shifts in spectra. The further main pedagogic goals of the experiment are that students will 1. Be able to interpret the 1H NMR data from both pure and substances in the mixture 2. Be able to process their own data with MNova software (or related ones) 3. Be able to make a decision on which signals in the 1H NMR spectra are appropriate for use in the quantitative determination and which are not 4. Have hands-on experience with NMR spectroscopy from preparing the sample, to measuring and processing data, and evaluating their results 5. Learn what kind of simplification of the 1H NMR spectrum is obtained after D2O exchange. Pharmacy students in an instrumental analytics lab course are second-year undergraduates. In their education, they completed courses in organic chemistry, basic analytical chemistry (mostly titrations and gravimetrical analysis), and basic inorganic chemistry. When performing this experiment, they were exposed to only 2 h of a theoretical seminar on NMR spectroscopy. Quantitative and qualitative determination using 1H NMR spectroscopy, directly in the formulation, is the very new approach for teaching NMR that we developed for pharmacy students in the instrumental analytics lab. There are certainly other approaches to analyze tablets quantitatively in the analytics lab course, such as LC−MS, HPLC, UV−vis spectroscopy, and titrations, and these methods are done and discussed in the lab course. According to students, the main advantages of NMR spectroscopy are (i) avoidance of expensive reference substances and (ii) a smaller time requirement than the other methods.7 For more advanced students this experiment might be directly combined and compared with HPLC quantification, but in our lab course it is not possible as the students learn the mentioned techniques in later lab courses. When using the mixtures only consisting of the OTC formulations, we are able to calculate absolute amounts of the components, not only their ratios. The complete sample as well as the sample used for the measurement has to be weighed and, from their ratios, absolute. Certainly, if pills are used or other impurities are present, without using an internal standard, only a relative ratio of the components can be determined. For achieving our pedagogic goal, deepening student understanding of NMR spectroscopy, this kind of result was satisfactory. However, for the adoption of this experimental setup for the more advanced students, we may suggest taking advantage of the standard addition method for the calibration (see additional information in the instructor guidelines in the Supporting Information) or using an internal standard.5,8 We suggest the use of maleic acid or 3-(trimethylsilyl)-1-propanesulfonic acidd6 sodium salt (DSS-d6).9 To increase the interest of the students, two- and threecomponent mixtures of pain relieving drugs were prepared using drugs that can be found in commonly marketed pain relieving tablets. In addition, two commercially available drug formulations were successfully analyzed in the direct, accurate, and rapid quantitative analysis without any further purification step in a 10 min analysis time. The compounds of interest are acetylsalicylic acid, paracetamol (acetaminophen), caffeine, and L-ascorbic acid, which are used as OTC drugs for pain relief (Figure 1).

Figure 1. Drugs used for creating two- and three-component mixtures for quantitative determination.

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EXPERIMENTAL SECTION

Materials and Instrumentation

A variety of low-field NMR instruments are on the market and are suitable for the use for this experiment. We used a Magritek Spinsolve NMR benchtop spectrometer (42.5 MHz) with standard 5 mm NMR-tubes, DMSO-d6, and D2O. We encourage students to acquire a 1H NMR spectrum of each possible component in DMSO-d6 with and without D2O and discuss the changes they observe. Prelab Exercise

For a successful experiment, it is important that the students are familiar with the basic theoretical background of 1H NMR spectroscopy. For quantification, it is essential that students understand how the integration and the number of protons measured can be used in determination of unknown concentrations. Questions can be found in the Supporting Information. Procedure

A group of two students obtained from the instructor a mixture and a tablet and were asked to make a qualitative determination of which components they have in their mixture and then to quantify them using 1H NMR spectroscopy. All of the single components were available for the students in the lab. The students were encouraged to acquire 1H NMR spectra of all the single components separately before and after D2O exchange and discuss the results obtained. The students dissolved the powdered mixture, approximately 20 mg in 0.6 mL of DMSO-d6, and acquired a 1H NMR spectrum. For the second 1H NMR spectrum, they added two drops of D2O. When tablets were used, the addition of D2O is recommended to complete dissolution of the samples (not more than 1.0 mL of total sample volume). In each case, a few discrete signals were used for quantification. Students need to determine themselves which signals are appropriate for the quantitative determination and whether the 1H NMR measurements before or after the exchange with deuterium should be used. It was essential to choose signals that do not exchange with D2O (as the integration is not as precise as by others), and do not overlap with other signals, and can be assigned to the correct number of protons of only one compound. The data obtained were put in a table similar to the one shown in Table 1. This assessment puts the students in the position to calculate the ratio of the substances in the mixture (for details see Supporting Information). B



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Table 1. Quantification from the 1H NMR Measurement of the Acetylsalicylic Acid/Paracetamol/Caffeine Tableta Compound Molecular weight (Mr) Integral height (I) Number of protons (P) that belong to integral (I) Ratio I/P Mass ratio (Mr × I/P) Mass ratio in % Actual mass ratios in a tablet in % a

Acetylsalicylic Acid, 250 mg

Paracetamol, 250 mg

Caffeine, 50 mg

180.16 2.50 3

151.16 2.00 2

194.19 0.52 3

0.83 149.53 44.8 45.45

1.00 151.16 45.2 45.45

0.17 33.01 9.9 9.10

See ref 10.

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HAZARDS DMSO-d6 is a combustible liquid that should be kept away from any source of ignition. D2O is hazardous if ingested. Acetylsalicylic acid is harmful if swallowed, and might cause mild skin irritation, serious eye irritation, and an allergic skin reaction. Caffeine is hazardous in the case of skin contact, and is an irritant to the eyes, ingestion, and inhalation. Paracetamol (acetaminophen) is hazardous in the case of skin contact, eye contact, ingestion, and inhalation. The students need to learn the hazards before working and need to check the safety data sheet for all the substances. The quantity used in each experiment is small compared to a typical recommended dose of the OTC medications. Nevertheless, consumption of large quantities should be avoided. Appropriate safety equipment (laboratory coat, safety gloves, and glasses) should be used by students at all times.

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Figure 2. 1H NMR measurements of paracetamol in DMSO-d6 (∼20 mg/mL) before (above) and after D2O exchange (below).

RESULTS AND DISCUSSION The first measurements done by students were those of the pure components in the mixtures. For illustration of these measurements, the 1H NMR spectra of paracetamol before and after deuterium exchange are shown in Figure 2. In those spectra, students observed that protons on heteroatoms are exchanged with D2O and that the NMR spectra were simplified. The students could determine with success which protons are exchangeable. The spectra of the other solitary components (before and after D2O exchange) can be found in the Supporting Information. Each group had a 5 h lab time for one two-component mixture, one three-component mixture, and one pill measurement. If less than 10% of one component is used, it was necessary to use more than 20 mg of the sample; approximately up to 50 mg could be used. The instructors should avoid such mixtures that contain less than 10% of one component or inform students that 50 mg of the mixture should be used for analysis. We show a quantitative determination of the twocomponent mixture of caffeine and paracetamol in a ratio 85%/ 15% (Figure 3). The quantitative analysis determination of a marketed product containing acetylsalicylic acid (250 mg, 45.45%)/ paracetamol (250 mg, 45.45%)/caffeine (50 mg, 9.10%) in one tablet done in the student lab is shown in Figure 4, and summarized in Table 1. The accompanying additives did not disturb our measurements of the homogenized tablet, making this method attractive for this kind of determination and not just for the mixtures of active substances. Another marketed mixture of acetylsalicylic acid and L-ascorbic acid was measured

Figure 3. 1H NMR measurement of a two-component mixture containing paracetamol (85%) and caffeine (15%).

accordingly and described in the Supporting Information. However, it should be stressed that with this method we determined the mass ratio of the components in a tablet, not their amount. If the total amount of active ingredients is known, the amount of each active substance can be calculated. The amount can also be determined using the standard addition calibration method.8 All of the measurements shown in the figures are performed by students. C



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of 1.88%, relative standard deviation of 1.87%, and confidence limit of 100.4 ± 0.55% (upper t probability of 99%) convinced us that the necessary accuracy and precision were achieved. The results of the measurements are normally distributed (the corresponding calculations are given in the instructor guidelines in the Supporting Information). This problem-oriented teaching method with hands-on experience resulted in measurable gains of student knowledge in 1H NMR spectroscopy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00105. Instructor guidelines (PDF, DOCX) Laboratory procedure for students (PDF, DOCX) 1 H NMR Spectra for instructors (PDF)

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Figure 4. 1H NMR measurement of a tablet containing acetylsalicylic acid (250 mg, 45.45%), paracetamol (250 mg, 45.45%), and caffeine (50 mg, 9.10%).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

All the details for the experiments, as well as some example measurements obtained in the student lab, are available in the Supporting Information. More than 50 two- and three-component mixtures have been analyzed, containing varying amounts of the substances from 10% to 90%. As an example, we showed the measurement of the sample containing caffeine (15%(m/m)) and paracetamol (85%(m/m)) (Figure 3). This experiment was done by 70 students attending an instrumental analytics course for pharmacy studies. The experiment itself, using different mixtures, has been performed more than 60 times in the student lab. This approach for qualitative and quantitative determination of OTC drugs created an attractive approach for learning NMR spectroscopy in the student lab. Our students enjoyed the experiment. Though other mixtures can also be used for teaching the basics in the NMR spectroscopy, pharmacy students find it attractive to use actual drugs for these kinds of experiments. The application of the active components attracts a lot of interest in our students. The experiment was evaluated in the way that the students gave feedback concerning their own experience. While describing their response, they used words (translated here into English) such as “exciting”, “own decision making”, “easier interpretation of the spectra”, and so on. Comparison of the student skills in a prelab assignments (see the Supporting Information), postlab questions, and exam clearly showed an improvement relative to all five defined pedagogic goals. Before introducing the NMR hands-on experiment, students (four previous semesters analyzed) were able to obtain 56% of the points available for the questions concerning NMR spectroscopy. In the two semesters after introducing this experiment, the total number of points our students scored in the NMR spectroscopy questions increased to the 77%. One of the prelab assignments was to determine the ratio of the components in the 1H NMR spectra the students obtained from the assistants. Less than 10% of the students were able to solve this problem without help. This number rose to 71% after successful performance of the experiment in their group. We applied different statistical tools and quantified accuracy (bias) and precision, in order to evaluate our student results. The calculated standard deviation

ORCID

Aleksandra Zivkovic: 0000-0001-5034-7916 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding of this project was kindly provided by the fund for teaching support (Lehrfö rderfonds) of Heinrich Heine University Düsseldorf, number L020401/15, as well as by the Serbian Ministry of Science and Technological Development, project number III43014.

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REFERENCES

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Quantitative Analysis of Multicomponent Mixtures of Over-the-Counter

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