Determining the Antifungal Agent Clioquinol by HPLC, the Not So

Feb 19, 2018 - To the best of the authors' knowledge, this represents the first time these components have appeared in this Journal as subjects of a q...
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Determining the Antifungal Agent Clioquinol by HPLC, the Not So Pure Preparation: A Laboratory-Based Case Study for an Instrumental Analytical Chemistry Course Peter M. Schaber* and Geoffrey Hobika Department of Chemistry and Biochemistry, Canisius College, Buffalo, New York 14208, United States S Supporting Information *

ABSTRACT: The case study approach provides students with a better appreciation of how scientists solve problems and conduct themselves in the “real world”. When applied to the undergraduate chemistry laboratory, this approach also challenges critical thinking skills and creativity in ways “cook book” experiments very often do not. This approach provides students the opportunity to learn analytical principles in the context of relevant everyday problems, stimulate a sustaining interest, and develop higher cognitive skills and reasoning abilities. The laboratory-based case study described here combines the use of small collaborative groups and a modern instrumental technique, with application to a common consumer product. Students are required to conduct themselves as chemists would, when addressing the problem of product purity claims, and to make a determination based on their experimental results that will be used to direct future action taken by a formulation company. This case study even addresses potential legal exposure issues the company may encounter with the Food and Drug Administration. In that context, students come to realize that a faulty conclusion could potentially have undesirable ramifications. In addition, students are provided with the opportunity to gain useful hands-on experience on instrumentation commonly used in modern analytical laboratories. A “professional” report must also be written by students in journal style format including all relevant data, calculations, and statistical and error analysis. Assessment was made using mixed methods. Results indicate the laboratory was largely successful in meeting its objectives. KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Laboratory Instruction, Collaborative/Cooperative Learning, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, HPLC, Instrumental Methods, Separation Science

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INTRODUCTION Several articles have recently been published in this Journal and others describing the use of various nontraditional instructional approaches.1−5 The case study approach is one that has proved successful not only at this institution6−10 but at others as well.11−16 In addition, a verity of articles have also appeared in the literature describing the use of modern instrumentation in the undergraduate laboratory, specifically high performance liquid chromatography (HPLC), as applied to the analysis of consumer products.17−25 Herein is described a laboratory-based case study, utilizing HPLC, with application to the separation of a common antifungal agent, clioquinol (5-chloro-7-iodo-8hydroxyquinoline), from its major impurity 5-chloro-8-hydroxyquinoline (Figure 1). To the best of the authors’ knowledge, this represents the first time these components have appeared in this Journal as subjects of a qualitative and quantitative determination laboratory. The laboratory-based case study approach is becoming ever more common in science-based courses because of the © XXXX American Chemical Society and Division of Chemical Education, Inc.

Figure 1. Structural formulas of compounds of interest in this experiment.

documented advantages offered, and the need for the implementation of more active learning and critical thinking initiatives at the undergraduate level.9,10,26,27 Compared to conventional laboratory experiments, the case study approach provides students with a better appreciation of how scientists actually conduct themselves and solve problems in the “real world”, and challenges student critical thinking skills and creativity in ways “cook book” experiments too often do not. Received: September 26, 2017 Revised: January 19, 2018

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laboratory. The compromise wavelength technique was used here since, in these laboratories, most students are very inexperienced with HPLC instrumentation at this point. Student groups are instructed to begin with an isocratic mobile phase consisting of 25% (0.050 M phosphoric acid)/75% methanol and are also informed that this mobile phase will result in excessively large k (retention factor) parameters that will need to be adjusted to an appropriate level (i.e., k = 1−5) prior to proceeding.30 They are advised that this objective can be accomplished by making stepwise adjustments in mobile phase composition, or by applying the method covered in the text31 using polarity index values. The instructor reminds students at this time that the instrument is equipped with a 5 μm octyldecylsilane (ODS, C18) reversed phase column. After identification of an appropriate mobile phase, students then are given a choice with respect to the method used for qualitative component identification (i.e., retention time vs spiking) and establishment of a calibration curve (i.e., variable injection size vs serial dilution), followed by collection, interpretation, and application of appropriate qualitative and quantitative data. (See Supporting Information for details.) Two weeks are given from the time the laboratory is first introduced to hand in the laboratory report. That report must be written in journal style format including an Abstract and Introduction, Theory, Experimental, and Results and Discussion sections. All relevant data, calculations, and error analysis must also be included. Materials to be handed in include the chromatographic method, relevant chromatograms upon which qualitative and quantitative analysis are based, and the least-squares calibration plot including the “best straight line” equation and correlation coefficient (r). The laboratory report must also address the question of company legal exposure based on statistical analysis at the 95% confidence level. A Data, Calculations, Error Analysis, and Results and Discussion Worksheet is provided not only to help student groups focus more on the required information to be included in the laboratory report, but also to serve as a template with respect to laboratory report order of presentation, making it somewhat easier for the instructor to grade multiple reports (see Supporting Information).

Well-designed laboratory-based case study experiments should to the greatest extent possible involve teamwork, and require students to collect, critically assess, and apply their experimental data to address the question(s) posed in the case. In addition, they should require a written report where there is a discussion of the significance of the results. A recent ACS symposium reviews the application of the case study method directly to analytical chemistry and the advantages it offers in this context,28 as does an example in the most recent literature.29 This approach provides students the opportunity to learn analytical principles in the context of relevant everyday problems, stimulate a sustaining interest, and develop higher cognitive skills and reasoning abilities. In this regard, the authors have identified the following series of learning objectives addressed in association with the performance of this laboratory-based case study experiment. Learning Objectives

1. Have students work successfully in small collaborative groups to address the “real world” problem of product purity claims. 2. Demonstrate increase student mastery of relevant material in a case study versus lecture setting. 3. Expose students to instrumentation commonly used in modern analytical laboratories (i.e., HPLC) and the acquisition of operational confidence via direct hands-on experience. 4. Have students groups use HPLC instrumentation while working independently, as a professional chemist would, with respect to appropriate method development and collection and interpretation of data. 5. Require student groups to make a determination, based on experimental results and statistical analysis, that will not only be used to direct the future action taken by a formulation company but also be used to address the issue of potential legal exposure (see The Case Study below). 6. Require the writing and submission for grading of a “professional” report, in journal style format, by student groups. 7. Evaluation of students’ experiences with respect to performing the laboratory. An unstated goal, but one that should not be overlooked in determinations of the type outlined, is the recognition of the importance of careful analytical study and interpretation of results. (Required equipment and materials for this laboratory can be found in the Supporting Information.) The laboratory instructor provides students with documents outlining the basic operation of the HPLC instrument, including an accompanying tutorial, along with the case, 1 week prior to the laboratory. Students are asked to familiarize themselves with these documents. During a 1 h prelaboratory session the instructor reviews the above materials emphasizing safety, provides helpful hints, and addresses questions. The instructor also reminds students that an important objective of chromatography is to achieve “baseline” separation of components in the minimum amount of time. Student groups need to consult the literature or online sources to determine a convenient detection wavelength in the UV (λ = 240−300 nm) for analysis of both components. Their choice needs to be approved by the instructor, and source(s) need to be identified prior to beginning the instrumentation portion of the

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THE CASE STUDY This experiment was inspired by an actual situation a regional Food and Drug Administration (FDA) laboratory was called in to address,32 modified to fit an industrial situation. The drug clioquinol (also known as 5-chloro-7-iodo-8-quinolinol; iodochlorhydroxyquin) is often prescribed as a topical treatment for fungal and bacterial infections of the skin. In its pharmaceutical form it has been shown to be very effective in treating patients. Typical preparations usually consist of hydrocortisone and clioquinol dispersed in an ointment or cream. Clioquinol is commercially prepared via the reaction of an aqueous solution of an alkali salt of 5-chloro-8-hydroxyquinoline with potassium iodide and a hypochlorite. A purification process follows to produce pharmaceutical grade product. Although the starting material itself possesses antifungal and antibacterial properties, it is much less effective than clioquinol and is often found as an impurity in the final product. The company in question is one that purchases pharmaceutical quality products from external sources and formulates the actual products sold. A concerning trend has recently been observed with respect to one of the company’s major products. A cream-based product containing clioquinol as the active ingredient, advertised to cure athlete’s foot, has precipitously B

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is appropriate and consistent with institution, state, federal, and local procedures.

lost a significant amount of market share. At a meeting focusing on the above issue, a procurement officer pointed out that in a money saving move the company recently switched their clioquinol supplier. This decision came against better judgment because the current source was notorious for inconsistent quality and lack of proper product purification procedures. The procurement officer added that the current overseas supplier quotes clioquinol purity at ≥99% with ≤1% 5-chloro-8hydroxyquinoline as the only significant impurity. Company chemists pointed out that although both clioquinol and 5chloro-8-hydroxyquinoline have antifungal and antibacterial activity, if the clioquinol is substantially contaminated with 5chloro-8-hydroxyquinoline, potency of their product could be adversely impacted. However, the chemists also pointed out that the potency issue may not be the company’s biggest problem. Company officials were reminded that the FDA only allows very limited variations from the level of active ingredient claimed on the label. If it is found that the variation is excessive, product seizure, recall, and significant fines for “truth in labeling” violations could be realized. Company chemists calculated that at the level of clioquinol contained in their product, at or above 2.5% contamination by the 5-chloro-8hydroxyquinoline impurity (i.e., ≤97.5% clioquinol purity), would trigger FDA action. The company quickly decided to have their quality control analysts determine the purity level of the most recent shipment of clioquinol form the supplier.

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RESULTS AND DISCUSSION This experiment was introduced and run for three consecutive years in the advanced instrumental analytical laboratory course. This course is largely populated by upper-level chemistry and biochemistry majors, many taking the course as an advanced elective. Several of the students have however had only minimal exposure with respect to hands-on experience operating sophisticated chemistry-based instrumentation. None of the students in this study had extensive previous experience operating HPLC instrumentation. The laboratory is proceeded by a 1 h in-class introduction (prelaboratory), immediately followed by a 3 h instrument demonstration and check-out session. The laboratory proper is typically run in an “open” fashion wherein each student group signs up for a 4 h time slot. This time is usually used for sample preparation and instrument operation. In these laboratories a compromise wavelength at λ = 248 nm was found to work very well. However, wavelengths in a range from 240 to 263 nm have also been successfully used. Students needed to decrease the polarity of the mobile phase to achieve “baseline” separation in minimal time. A mobile phase consisting of 5% (0.050 M phosphoric acid)/95% methanol gave the best results (Figure 2) with respect to combined column resolution (Rs) and k values.30

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STUDENT ACTION Small student groups (typically 3 students) play the role of quality control analysts working for a pharmaceutical formulation company. Their first task is to develop an HPLC method for the “baseline” separation (in minimum time) of the two components in question (i.e., clioquinol and 5-chloro-8hydroxyquinoline), and the qualitative identification of each. They are then provided with a sample of clioquinol recently obtained from the supplier under suspicion. Student groups are next required to quantitatively determine the % purity of clioquinol in the supplier’s sample. On the basis of statistical analysis (at the 95% confidence level), students are also required to determine if the company is in danger of legal exposure with respect to the FDA’s “truth in labeling” statutes.

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HAZARDS Appropriate gloves, protective clothing, and safety goggles must be worn at all times during the performance of this experiment. Clioquinol and 5-chloro-8-hydroxyquinoline can cause irritation to the eyes and skin on contact and to the respiratory system if inhaled. It should be noted that the toxicological properties of 5-chloro-8-hydroxyquinoline have not been fully investigated, and that clioquinol is a neurotoxin in large doses. Methanol is a known poisonous substance via ingestion and can cause blindness and death. Inhalation of vapors can irritate mucous membrane and the respiratory tract and aggravate the central nervous system. Methanol is also flammable, so no open flames or sparks should be present. Concentrated solutions of phosphoric acid are also irritating to the skin and mucous membranes. It is advisible to perform manipulations with all these substances, whenever possible, in a fume hood. When filtering solutions using a 0.45 μm disk, ensure that the filter disk is securely attached to the syringe lock mechanism prior to filling, and avoid overfilling of the syringe to prevent leakage and exposure. All waste should be disposed of in a manner that

Figure 2. Sample chromatogram showing the separation of clioquinol from 5-chloro-8-hydroxyquinoline.

A mobile phase consisting of 20% (0.050 M phosphoric acid)/80% methanol did result in a marginally acceptable k value for clioquinol. However, the Rs value is very much larger than it needs to be to achieve “baseline” separation in minimal time. Linear clioquinol calibration curves were constructed by plotting peak area versus concentration over the range 1.5 × 10−4 to 6.0 × 10−4 g/mL. Correlation coefficients (r) of >0.99 are typical for the “best straight line” (see Supporting Information). All student teams were able to complete the experiment, as designed, in the single 4 h time slot. Assessment was made using mixed methods:33 laboratory report results, comparison of laboratory report score versus first C

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participation of group members, with respect to student interactions. Student reactions to HPLC instrumentation were also very positive (see Supporting Information). An important objective of the Not So Pure Preparation case study was for students to work as independently as possible in the laboratory, and as much as possible like the way a professional chemist does in the “real world”. A substantial majority of the students agreed that they did indeed perceive themselves as working independently as chemists do, were confident in their ability to use HPLC instrumentation in the future as a research investigator, and expressed a feeling of increased responsibility for instrument operation and data collection. In addition, students with both previous or even very limited exposure to instrumentation experienced an increase in their self-efficacy for working with the technology. Limited experience with instrumentation, however, did tend to stimulate more student interest, a feeling of understanding, and a stronger desire to use HPLC instrumentation in the future.

in-class test scores, and administration of a student survey instrument. It must be recognized, however, that although the results indicate this laboratory-based case study was largely successful in meeting its objectives, the relatively small sample size (n = 15) must be taken into consideration. The quality of the reports did of course vary (see Supporting Information); however, an average grade of 80% (B in current grading scheme) was achieved. (These results must be considered in the context that this experiment was the first introduced in the laboratory schedule and most students were new to the journal style format for laboratory reports.) In addition, all student groups able to correctly complete the analysis and statistical portions of the laboratory were also able to correctly make the appropriate determination with respect to potential company legal exposure. These results accomplished two major objectives of the laboratory report, per se. One student group did however made an error in their calculations that resulted in a faulty conclusion. However, when the laboratory report was graded and handed back to the students, that group quickly recognized the error and realized that their faulty conclusion could potentially have had undesirable ramifications if such occurred in the “real world”. This not only resulted in a “teachable moment” but also accomplished an unstated objective of the experiment, that is, the realization that an analysis such as those performed in this study can very well have a profound impact on others. In addition, most unknown samples used in this experiment were contaminated with 5chloro-8-hydroxyquinoline at a level resulting in a definitive conclusions. However, one unknown was contaminated at a relatively low level resulting in a more nebulous situation requiring additional critical thought by the students. This situation resulted in another “teachable moment” wherein a discussion ensued relating to the need for the collection of additional data so that a good estimate of standard deviation (σ) could be made, and the potential arrival at a more definitive conclusion. All students took both the lecture and laboratory sections of the course. The first test in the lecture section covered basic statistics (calculation of standard deviation, calibration plots, confidence intervals, etc.), and an introduction to chromatography (calculation of k, Rs parameters, etc.) and liquid chromatography (including HPLC, mobile phase polarity adjustment, etc.). These are the same basic principles necessary for the successful completion of this case study. When comparing the laboratory report scores versus test scores, in each of the three semesters the laboratory was run, it was noted that laboratory report grades were significantly higher than either the average or median test scores (see Supporting Information). A student survey assessment tool, designed in part to evaluate the students’ experiences9 with respect to performing this laboratory-based case study, was also administered to all students taking the course. The general goal of the survey was to evaluate the extent to which students’ experiences, upon exposure to a “real world” situation, working in small groups and using HPLC instrumentation, increased their view of chemistry as relevant to the real world, developed their critical thinking skills and confidence for instrumentation operation, and increased their enjoyment of chemistry, etc. In short, the Not So Pure Preparation case study elicited extremely positive reactions from the students, especially with respect to understanding phenomena, being more interesting, remembering more, and eliciting a keen sense of responsibility for full

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CONCLUSION

The Not So Pure Preparation is a laboratory-based case study that combines the use of small collaborative student groups and a modern instrumental technique, with application to a common consumer product. Without the usual close supervision by the instructor, student groups were able to master the ability to operate HPLC instrumentation in a more independent manner than otherwise. This was due to the fact that they were required to work largely without the normal close supervision of the instructor, and had to rely on each other more so than in a non-case-study scenario. This allowed students to demonstrate to themselves the ability, for the most part, to independently become more fully familiarized with the instrumentation per se and to understand more in-depth the theory underlying HPLC functionality and operation. Along the way, students were able to successfully select an appropriate compromise wavelength for component detection, develop a method via mobile phase polarity adjustment to achieve “baseline” separation with reasonable k and Rs values, qualitatively identify the components of interest, and, through the use of external standards, quantitatively determine the % concentration of clioquinol in an unknown sample via application of a linear least-squares plot. Statistical application of data collected allowed students further to make a determination, at the 95% confidence level, and apply that knowledge to solve a “real-world”-based problem. With respect to the laboratory report, all student groups were able to hand in a “professional” report in journal style format. Assessment data supports the indication that this laboratory was largely successful with respect to student’s mastery of relevant material being better in a case study, coupled with a group learning environment, versus lecture. Collected data also supports the importance of introducing instrumental methods, such as HPLC, at an early stage in a student’s career especially to students of limited experience. In summation, findings suggest that the Not So Pure Preparation was largely successful in accomplishing its objectives, and that use of HPLC technology coupled with a group learning approach produced a very successful and enjoyable student learning experience. D

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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.7b00750. Instructor notes containing detailed experimental procedure and guidelines, sample student data, CAS registry numbers for chemicals used, assessment instruments, summary and complete assessment details, and analysis of survey results (PDF, DOCX) Student handout (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Peter M. Schaber: 0000-0003-2232-8595 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank the Canisius College Earning Excellence Program (CEEP) for their generous support and the students of CHM 430 L, especially Jena Congilo, Austin Gilbert, and Samantha Caicos, for their participation in this project, and for generation of the data necessary for completion of this project. Special thanks also goes out to Valerie Banks for her valuable technical assistance.

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