Greener Chromatographic Approaches for Dissolution Testing of Solid

Research Article pubs.acs.org/journal/ascecg

Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Greener Chromatographic Approaches for Dissolution Testing of Solid Pharmaceutical Formulations Adam Socia,*,† Yong Liu,*,† Xiaoyi Gong,‡ Orane White,‡ Andreas Abend,† and W. Peter Wuelfing† †

Analytical Sciences, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States Analytical Commercialization and Technology, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States

‡

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S Supporting Information *

ABSTRACT: Dissolution testing is a critical enabler for formulation process development as well as determination of product quality at release and on stability for solid oral dosage forms. In the pharmaceutical industry worldwide, this type of test is performed in the range of millions annually. We developed an improved chromatographic protocol combining the utilization of smaller internal diameter (i.d.) columns, superficially porous column technology, injection cycle time for gradient re-equilibration, system dwell volume understanding, and basic separation concepts for optimization as a greener, faster, yet robust way to conduct dissolution testing. Comparing this approach to standard analysis using a conventional approach, this methodology provides 70−80% reduction in solvent consumption and waste generation, as well as run times with equivalent accuracy, precision, and robustness. Feasibility of this approach was demonstrated by applying it to multiple drug products and those head-to-head comparisons showed that dissolution profiles and overall variability are comparable to those obtained by the conventional chromatographic approaches. KEYWORDS: (Ultra) High performance liquid chromatography, Green chromatography, Dissolution testing, Pharmaceutical analysis

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use.7−11 In fact, HPLC is the most important and broadly used analytical technique in pharmaceutical laboratories.12 When the use of an off-line analysis technique is employed (i.e., HPLC), the method should be optimized in order to generate the least amount of hydro-organic solvent waste possible, while maintaining a high level of robustness, accuracy, and ease of use. Solvent waste reduction by the application of smaller diameter columns on conventional HPLC or UHPLC, or even microfluidic and capillary HPLC, has been implemented for analysis of active pharmaceutical ingredients (API) and synthetic intermediates.4,13 However, little work of “greening” chromatography has been reported or emphasized in dissolution testing of a formulated drug product. Conventional HPLC analyses of dissolution samples use 4.6 mm i.d. columns and flow rates between 1.0 and 5.0 mL/min. Since a typical dissolution study, depending on the purpose of the data, is carried out by analyzing 3−12 replicates in dissolution media at 6−8 different time points to generate a profile, complete analysis of all the samples from a dissolution experiment can take several hours, resulting in hundreds of milliliters of hydro-

INTRODUCTION AND THEORY Dissolution tests are routinely employed in both research and development laboratories and manufacturing quality control facilities of solid dosage forms in pharmaceutical industry. As an important analysis,1 it is used routinely to assess product quality at release, and on stability, guide formulation development and may be used as a predictor of product pharmacokinetic performance (e.g., in vivo/in vitro correlation [IVIVC]). According to data compiled from the Food and Drug Administration (FDA),2,3 there are currently 1317 filed products that employ dissolution testing, and of those, the vast majority use off-line analysis by liquid chromatography. Green analytical chemistry represents an important part of green chemistry efforts in research laboratories and manufacturing quality control facilities, with a focus on reducing hydro-organic solvent waste in analytical experiments.4−6 Though savings can be modest, when multiplied by a greater number of individual analytical chemists and tests performed, it can have a significant cumulative impact. Although there has been good effort in using UV-probes and other in situ detection techniques in dissolution testing, the vast majority of dissolution testing still uses high-performance liquid chromatography (HPLC) for sample analysis because of its specificity, robustness, high reproducibility, excellent accuracy, and ease of © XXXX American Chemical Society

Received: August 28, 2018 Revised: November 2, 2018 Published: November 5, 2018 A

DOI: 10.1021/acssuschemeng.8b04311 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

grade deionized water was used for preparation of dissolution media, mobile phase, and diluent solutions. Janumet (50 mg of sitagliptin/ 500 mg of metformin, Lot# N019913) and Actoplusmet (15 mg of pioglitazone/500 mg of metformin, Lot# A23990) tablets were purchased through Myoderm (Norristown, PA), and ruzasvir/ uprifosbuvir (80 mg/225 mg) developmental tablets were produced internally (Merck & Co., Inc., West Point, PA, USA). Instrumental Parameters. An Agilent 1290 Infinity Ultra Performance Liquid Chromatography system with a quaternary pump (Santa Clara, CA) and an Agilent 1100 High-Performance Liquid Chromatography system with a binary pump were used. Both systems had photodiode array detectors. Data acquisition and processing were performed using Waters EMPOWER3 chromatography software. Sample and Standard Preparations. Since the intent of this experimental design was to demonstrate that optimized methods are not only “greener” than filed ones, but that the resulting data were equivalent in terms of precision and accuracy, and that many USP monographs specified a single time point only, time points were selected here to produce dissolution curves that would be relevant to a compound in development and would allow multiple points of comparison between the filed and “green” methods. The Actoplusmet tablet dissolution samples were generated as per its USP monograph,16 using 6 replicate vessel equivalents per formulation. Samples were withdrawn at 0, 10, 15, 20, 30, 45, and 60 min and analyzed. Standards were prepared as per the USP monograph. The Janumet tablet dissolution samples were generated using the USP apparatus II1 with 900 mL of 0.025 M NaCl dissolution media at 37 °C. Six replicate vessels per formulation were used, and samples were withdrawn at 0, 10, 15, 20, 30, 45, and 60 min and analyzed. Standards were prepared at approximately 0.072 mg/mL sitagliptin and 0.56 mg/mL metformin in 50 mM potassium phosphate solution:acetonitrile (3:1). Ruzasvir/uprifosbuvir tablet dissolution samples were generated using the USP apparatus I1 with 900 mL of 0.3% (w/v) SDS in 20 mM pH 6.8 sodium phosphate buffer dissolution media at 37 °C. Six replicate vessels per formulation were used, and samples were withdrawn at 0, 5, 10, 15, 20, 25, 30, 45, and 60 min and analyzed. The ruzasvir/uprifosbuvir tablet dissolution media is from the method filed from our team for the release and stability testing for the Phase II clinical batches. The program was terminated; therefore, it was not defined by the FDA. Standards were prepared at approximately 0.1 mg/mL ruzasvir and 0.25 mg/mL uprifosbuvir in 1:1 (v/v) ACN:20 mM pH 4.0 acetate buffer. Doravirine standards were prepared at approximately 0.12 mg/mL in 3:2 (v/v) acetonitrile:water. Experimental Design. Using the standard and sample solutions prepared above, the chromatographic separations were carried out in accordance with the applicable original methods, using either USP monographs (Actoplusmet) or methods used during internal drug product development (Janumet, ruzasvir/uprifosbuvir, and doravirine) [Table S1]. These methods all utilized columns that had an internal diameter of 4.6 mm and lengths of 50 mm or 150 mm. The sequence run time was recorded and multiplied by the flow rate to determine total waste effluent generated per sequence. Analyses were performed using the Agilent 1100 HPLC system, and an instrument cycle time was noted to be on average approximately 1.4 min. All sample and standard solutions were then transferred onto the Agilent 1290 UHPLC system and run using the modified “greener” developmental parameters shown in Table S1. Parameters that were purposefully modified were a combination of the reduction of the column internal diameter along with (1) modification of mobile phase strength and/or (2) gradient program and/or (3) flow rate. Columns of a similar stationary phase type to the original analyses were chosen. As a result of these method modifications, the injection volumes and the analysis run times were adjusted accordingly. Variables, such as detection wavelength, column temperature, and analytical concentrations, were unaltered. The sequence run times were recorded and multiplied by the flow rates to determine total waste effluent

organic consumption and effluent waste per analysis. Within our company alone, we estimated over 800 and 2000 dissolutions in the development and commercialization stages, respectively. Therefore, using this approach we outline here could theoretically save ∼900 L of hydro-organic waste from being generated and disposed per year (2800 dissolution tests × 400 mL waste per test × 80% reduction). In the manufacture supply phase, 10 000 dissolution analyses are performed each year, on the basis of typical needs for the commercial testing metrics from our five highest selling solid dosage form products.14 In contrast to the practice in development and commercialization, one time point measurement of dissolution is generally performed in this stage. The hydro-organic waste generated is estimated to be 1200 L (10 000 dissolution tests × 150 mL waste per test × 80% reduction). Overall, the estimated hydro-organic waste for dissolution measurement at Merck is about 2100 L. Assuming the effluent waste contains 30% acetonitrile, that saves approximately 600 L of acetonitrile from being purchased, transported, and consumed, and at a cost of approximately $170 per liter,15 that is a savings of almost $110 000 per year, even without factoring in waste disposal cost savings and the environmental impact. Therefore, even though solvent waste reduction can be modest from a single analytical experiment, when multiplied by the number of samples needing to be analyzed per experiment, the number of experiments performed in an analytical laboratory, and the number of analytical laboratories across the industry, it will have a significant cumulative positive impact when greener analytical techniques are broadly employed in the pharmaceutical industry for dissolution testing. In this study, we report a comprehensive chromatographic approach to reduce the hydro-organic waste in HPLC analysis, and thus “greening” dissolution testing. The feasibility of the green analysis approach is demonstrated on challenging combination drug products, such as ruzasvir/uprifosbuvir, sitagliptin/metformin (Janumet), and pioglitazone/metformin (Actoplusmet) tablets. Additional analyses were performed using doravirine for dissolution method optimization studies, further “greening” the method. It is not the author’s intent to replace existing methods with the greened ones detailed here, but rather use these as examples in order to show that equivalent analytical results can be obtained via the optimization of chromatographic parameters, and that these techniques should be employed as early in method development as possible to achieve the greatest impact to sustainability. It is also not the author’s intent here to redevelop the methods to use greener substitutes for the existing solution compositions, such as replacing acetonitrile and phosphate salts with ethanol and carbonate salts.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Metformin hydrochloride drug substance was purchased from the USP (Lot R069H0, Rockville, MD). Pioglitazone drug substance was purchased from Sigma-Aldrich (Allentown, PA) and was 99+% pure. Sitagliptin phosphate, ruzasvir, uprifosbuvir, and doravirine drug substances were internally provided (Merck & Co., Inc., West Point, PA, USA) and were 99+% pure. Mass-spectrometry grade acetonitrile, potassium phosphate monobasic, 85% phosphoric acid, sodium phosphate dibasic anhydrous, sodium dodecyl sulfate, sodium acetate trihydrate, glacial acetic acid, citric acid, sodium chloride, sodium phosphate monobasic monohydrate, and sodium hydroxide were purchased from Fisher Scientific (Pittsburgh, PA) and were all ACS grade or better. In-house USPB


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ACS Sustainable Chemistry & Engineering generated per sequence. An instrument cycle time was noted to be on average approximately 1.1 min for the Agilent 1290 UHPLC system.

Table 1. Comparison of Total Waste Generated and % Reduction for Hypothetical Analyses Run with 4.6 mm i.d. (Original), 3.0 mm i.d., and 2.1 mm i.d. Columnsa

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RESULTS AND DISCUSSION In general, for a dissolution testing, an n = 6 vessel dissolution analysis with samples taken at 7 time points for analysis results in 57 HPLC injections (ninj), including vessel blanks and standard injections. If the HPLC analysis is performed using typical conventional method conditions, including a column with a 4.6 mm i.d. (radius [r1] = 2.3 mm), the flow rate ( f1) at 1.0 mL/min, and the run time per injection (ti) at 3 min, the volume of effluent waste generated (V) for the complete dissolution analysis is approximately 171 mL, as calculated by eq 1. By reducing the column i.d. to 3.0 mm (r2 = 1.5 mm) while using the same stationary phase and column length, an equivalent analysis (ti = 3 min) can be achieved with a reduced flow of 0.43 mL/min, maintaining the same linear velocity as calculated by eq 2,17,18 resulting in a waste volume of only 73 mL, a 57% reduction from that of the original analysis. Further lowering the column i.d. to 2.1 mm and an equivalent flow rate of 0.21 mL/min will generate only 36 mL of waste effluent, a reduction of nearly 80% from that of the original analysis. V = ft i n inj

ij r yz f2 = f1 jjj 2 zzz jr z k 1{

% reduction in volume from original

effluent volume with cycle time (mL)

% reduction in volume from original

4.6 mm i.d. 3.0 mm i.d. 2.1 mm i.d.

171 73 36

57 79

251 105 53

58 79

a

Analysis time (ti) of 3 min and an instrument cycle time (tc) of 1.4 min.

Increasing the flow rate is a simple way to reduce the run time and increase throughput, but may generate more waste as a result of flow rate increase. Using a 2.1 mm i.d. column, the above method parameters, with the same capacity factor (k′ = 3.0; eq 5), where tR is the retention time of the analyte, and t0 is the retention time of a unretained component, various flow rates can be employed to reduce the analysis time (ti), determined by eq 5. The volume of effluent waste can be recalculated by eq 3 and compared to that of the original analysis [Figures 1 and 2]. t − t0 k′ = R t0 (4)

(1)

2

(2)

t i = t R + 1.7t0

In practice, however, there are several important factors that are not included in the straightforward equations above for calculating waste volume. Most importantly, the instrument cycle time (tc), which is the fixed amount of time that passes from the end of data collection for an injection until the injection of a subsequent sample, is missing from the equations mentioned above. This variable includes the mechanical and electronic steps to prepare the instrument for analysis, such as auto sampler arm movement, injector needle washing, filling and injecting, as well as detector signal balancing. Instrument cycle time is critical to waste volume calculations, as the mobile phase continues to flow during this time, resulting in generation of additional waste. As the HPLC analysis run time for each injection decreases, the impact of the instrument cycle time increases. Adding the instrument cycle time value to eq 1 results in eq 3. V = f ((t ininj) + (tc(ninj − 1)))

column i.d.

effluent volume without cycle time (mL)

(5)

Clearly there is still a significant reduction in waste generated and a significant reduction in run time when using faster flow rates than the direct scaled down value. For example, when using a flow rate of 0.21 mL/min, there is a ∼80% reduction in waste and no reduction in analysis time, vs a ∼ 5% reduction in both waste and run time at 1.0 mL/min. Practically, higher flow rate will result in elevated back pressure on conventional packed columns. This can prevent the utilization of this approach on existing HPLC instruments, which are still being used in many quality control laboratories, such as Agilent 1100 systems. One way to circumvent this is to employ columns that contain 4−5 μm superficially porous particles (SPP). These columns provide similar efficiencies as ∼3 μm particle columns but with a much lower backpressure near that of a 5 μm particle. Since dissolution usually only requires the separation of the active(s) from the void, and each other in the case of multiple actives, ultrahigh efficiencies are not as important as when a stability indicating method is developed. However, columns with higher efficiencies generate sharper chromatographic peaks, which can allow lower injection volumes to be used, reducing the instrument cycle time and increasing the greenness of the separation further, making these SPP columns a better choice than a traditional 5 μm particle column for older HPLC systems with pressure limitations. Additionally, many methods are developed with excessive retention and resolution, and their run times can be reduced through optimization of chromatographic gradient and/or the solvent strength by increasing such things as the as salt additive and/or organic modifier concentrations. Lastly, dwell time can be used for column equilibration to further reduce total run time. These approaches are illustrated in the examples below. Doravirine. The most straightforward comparison of the reduction in effluent waste and sequence analysis time between the original and greened methods is from doravirine. For these

(3)

Using an instrument cycle time of 1.4 min and the prior data, the original analysis would now generate approximately 250 mL of waste, a 32% increase from not including the cycle time. A comparison of the total waste volume and % reduction with and without instrument cycle time is shown in Table 1. Experimentally, the use of our UHPLC system reduced the instrument cycle time from 1.4 to 1.1 min, which is a ∼20% reduction. While the reduction of the column i.d. and subsequent scaling of the flow rate can yield a clear and significant waste reduction,13,19 this scaled-down method still has the same sequence analysis time. By decreasing the sequence analysis time, the overall throughput of the lab is improved. This results in fewer instruments needing to be in-use at a given time, requiring less electricity to be used per analysis and a smaller lab footprint, which is another important perspective for greening analytical chemistry.19 C


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Figure 1. Comparison of total sequence analysis time (n = 57 injections) and total waste generated vs flow rate for hypothetical analyses run with 4.6 mm i.d. (original) and 2.1 mm i.d. (green) columns. Analyte retention time (tR) is based on a capacity factor (k′) of 3.0, an injection run time of tR+1.7t0, and an instrument cycle time (tc) of 1.4 min.

Figure 2. Comparison of % reductions from original analysis for sequence analysis time (n = 57 injections) and total waste generated vs flow rate based on data from Figure 1.

resolved from the void is k′ > 2,19,20 this method represents the minimum separation allowed. This resulted in a calculated 43% shorter sequence analysis time and 88% reduction in effluent waste compared to the filed method [Table 2]. Janumet. For these analyses, the same column type and length were used, and the only significant changes were to the internal diameter of the column and the modification of the mobile phase ratio. The flow rate was scaled down from 2.0 mL/min for the 4.6 mm i.d. column (used for commercial product) to 0.45 mL/min (slightly faster than that from eq 2) and 1.35 mL/min for the 2.0 mm i.d. column, separately. Method development was performed on the UHPLC analyses, focusing on the concentration of potassium ions and the level of acetonitrile in the method in order to again reduce the k′ from 5 to 2 for sitagliptin while maintaining a resolution of 2. It was observed that increasing the potassium concentration decreased the retention times of both sitagliptin and metformin, and increasing the acetonitrile content increased the resolution of the separation, affecting the sitagliptin to a greater extent than the metformin. Therefore, 100 mM potassium phosphate:acetonitrile (3:2) was used for the developmental green analyses, shown in Table S1, as the

analyses, the same column type, length, and mobile phases were used, and the only significant changes were the internal diameter of the column. The flow rate was scaled down proportionally in accordance with eq 2 from 1.5 mL/min for the 4.6 mm i.d. column to 0.31 mL/min for the 2.1 mm i.d. column. Even though the method is a gradient, the elution of the doravirine peak occurs in the isocratic portion (40% acetonitrile), with the gradient being used to wash off nonanalyte peaks from the column. According to Table 2, the calculated reduction in effluent waste was 81%, which was in-line with the predicted value of 79% from Table 1. Additionally, there was a slight decrease in the calculated sequence analysis time of 7%, owing to the difference in injection cycle time between the UHPLC and HPLC systems (1.1 vs 1.4 min). For the comparison using the modified developmental doravirine “DEV2” method [Table S1], the isocratic portion of the gradient was raised from 40% acetonitrile to 50% acetonitrile. This was done to optimize the sequence analysis time of the method by reducing the retention time of doravirine so that k′ ∼ 2 instead of k′ ∼ 5. Since recommended values for separations to ensure that the peak of interest is wellD


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ACS Sustainable Chemistry & Engineering Table 2. Solvent and Sequence Run Time Reductions for Each Method Set Evaluateda,b compound and method doravirine filed (k = 4.2) doravirine DEV1 (k = 5) doravirine DEV2 (k = 2) Janumet USP Janumet DEV2 Janumet DEV1 Actoplusmet USP Actoplusmet DEV1 Actoplusmet DEV2 diphenhydramine in-house diphenhydramine DEV1 (k = 1.8) diphenhydramine DEV2 (k = 0.5) (matches original k′) ruzasvir/uprifosbuvir FILED ruzasvir/uprifosbuvir DEV

column (cost in $) Waters Atlantis T3 (50 mm × 4.6 mm, 5 μm) ($490) Waters Atlantis T3 (50 mm × 2.1 mm, 3 μm) ($500) Phenomenex Luna SCX (50 × 4.6 mm, 3 μm) ($570) Phenomenex Luna SCX (50 × 2.0 mm, 5 μm) ($570) YMC pack ODS AM (150 × 4.6 mm, 3 μm) ($645) Agilent Poroshell 120 SBC18 (50 × 2.1 mm, 2.7 μm) ($411) Phenomenex Onyx Monolithic C18 (50 × 4.6 mm) ($835) Agilent Poroshell Phenyl-hexyl (50 × 2.1 mm, 2.7 μm) ($411)

GL Sciences Inertsil ODS-3 (50 × 4.6 mm, 3.0 μm) ($428) Agilent Poroshell 120 SBC18 (50 × 2.1 mm, 2.7 μm) ($411)

sequence analysis effluent waste (mL)

% reduction in effluent waste from original

353 (calc) 68 (calc) 42 (calc)

sequence analysis time (hrs)

% reduction in sequence analysis time from original

3.9 81 88

605

3.6 2.2

7 43

5.0

121 66 414

80 89

1.5 2.4 6.9

70 52

118 87 280 (calc)

71 79

2.0 2.9 2.0

71 58

49 (calc)

83

493

0

4.1

134

73

1.5

63

a

Doravirine sequence analysis times and waste generation are calculated based on method optimization chromatographic data and the subsequent application of eqs 3 and 5 for analysis of a full sequence. Column cost taken from vendor websites. bNote: DEV1 and DEV2 methods represent developmentally modified methods used to test different chromatographic parameters and are detailed in Table S1.

Figure 3. Comparison of dissolution profiles for sitagliptin in Janumet obtained by the 3 different methods detailed in Table S1.

analyses, the slower flow “DEV1” generates 45% less waste at a cost of a 60% longer sequence run time, although the absolute difference in analysis time is less than an hour. Regardless of the method used, the data (Figure 3) were comparable in terms of dissolution profile, with a “dissolution similarity factor” (F2)21 greater than 70. Actoplusmet. In order to explore combination product dissolution, and the application of faster flow rates and shorter sequence analysis times when using smaller internal diameter columns, Actoplusmet was selected as a test compound. The USP method calls for the use of an 150 mm column, but for optimization, a 50 mm column was used. Flow rates that were greater than those calculated from a direct scale down based on changes in internal diameter were used, generating faster

buffer concentration was increased as part of the method optimization to reduce chromatographic analysis time while maintaining equivalent analytical results. The full sequence was then run on the three different methods, and the sequence analysis time and effluent waste were recorded. According to Table 2, the actual reduction in effluent waste for the higher flow rate “DEV2” method was 80%, which was in-line with the predicted value of 79% from Table 1, and a significant decrease in the calculated sequence analysis time of 70%. Similarly, the slower flow rate “DEV1” method showed an 89% decrease in effluent waste and a 52% reduction in sequence analysis time due to the optimization of k′ and shortening of the injection run time from 5 to 1.5 min. However, when comparing the optimized “DEV1” to “DEV2” E


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Figure 4. Comparison of chromatograms using a 4.6 mm i.d. column (USP) and 2.1 mm i.d. column (DEV 1 and DEV 2) on UHPLC systems for Actoplusmet analysis. Note that the citrate peak is from the dissolution media.

analysis times and slightly more waste. An SPP column of similar stationary phase (Agilent Poroshell 120 SB-C18) was also used in place of the method column in order to establish the generation of a dissolution platform for future testing (Table S1). As shown in Figure 4, there are three peaks seen in the sample chromatograms, the late eluting and small pioglitazone, the large metformin, and an early eluting citrate peak, arising from the buffer salt used to make up dissolution media. Most impressively, when using the greened method at 1.0 mL/min flow rates (DEV 1), the full elution of all peaks occurs before the amount of time it would take for elution of the citrate peak when using the USP method. Even using a slower flow rate of 0.5 mL/min (“DEV2”), the later eluting pioglitazone elutes near to the citrate peak in the USP method. Overall, the comparison for this experiment showed a 71% reduction in effluent waste and sequence analysis time when using the faster flow rate (DEV 1) and a 79% reduction in waste/58% reduction in run time at the slower flow rate (DEV 2). Additionally, the use of the superficially porous Poroshell columns allows for high flow rates that are still highly efficient and a low enough backpressure suitable for Agilent 1100 systems with lower pressure limits.22 This is of course provided that the cycle times are sufficient to re-equilibrate the column. Ruzasvir/uprifosbuvir. The filed method for the analysis of ruzasvir/uprifosbuvir is a case where the analytes elute during the gradient portion of the analysis. The gradient programs for the filed and optimized methods are shown in Table 3. When optimizing methods where the analyte is eluted during the gradient, it is important to understand the effect of the system dwell volume23 on the separation, specifically, what ratio of mobile phases is at the head of the column at what time. By adjusting the program gradient time by adding the dwell time (tD), calculated in eq 6, to the program time, this information can be obtained. In our case, the UHPLC system had a dwell volume (Vd) of 0.52 mL. tD =

Vd F

Table 3. Comparison of ruzasvir/uprifosbuvir Gradient Programs for 4.6 mm i.d. (Filed; Left) and 2.1 mm i.d. (“Greened”; Right) Methodsa program time (min)

dwell time adjusted program time

%A

%B

0.0 1.5 1.6 2.0 2.1 3.0

0.0 0.3 1.8 1.9 2.3 2.4 3.0

90 90 25 10 10 90 90

10 10 75 90 90 10 10

program time (min)

dwell time adjusted program time

%A

%B

0.0 0.10 0.11 0.13 0.50

0.0 0.35 0.45 0.46 0.48 0.50

90 90 10 10 90 90

10 10 90 90 10 10

a

The UHPLC dwell volume (Vd) was previously determined to be 0.52 mL, and the dwell time (TD) is calculated by eq 6 and added to the program time to obtain the adjusted times. Note that the TD for the greened method is slightly larger than that of the filed method (0.35 vs 0.3 min) due to the slower flow rate (1.5 vs 2.0 mL/min).

significant wasted chromatographic space in the filed method immediately following the elution of the ruzasvir, shown in Figure 5. Wasted space in the chromatogram results in more effluent waste being generated and a longer than necessary analysis time. Therefore, the method was “greened” by not only replacing the use of a smaller internal diameter column ,as before, but also removing the initial hold time, so that the gradient begins as close to the elution of ruzasvir as possible. Additionally, since the gradient is used to elute compounds that are not chromatographed, also known as a “wash-off”, it is important to maintain the high organic content at the end. However, if the injection run time is set to end close to the maximum solvent elution strength reached, the re-equilibration can be achieved by exploiting the cycle time. At high enough flow rates, such as 1.5 mL/min, a cycle time of 1.1 min will pass at least 15 column volumes, which can be used for the reequilibration when using a 50 mm × 2.1 mm i.d. column, which is more than enough for reproducible and stable chromatography. Again, the use of the superficially porous Poroshell columns allows for high flow rates that are able to be run on the lower limit Agilent 1100 systems, provided that the dwell volume

(6)

The dwell time can be used to ones advantage in developing a greener method by several factors. First, once the dwell time adjusted gradient is determined, it is clear that there is a F


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Figure 5. Comparison of chromatograms using a 4.6 mm i.d. column (filed) and 2.1 mm i.d. column (greened) on UHPLC systems for uprifosbuvir/ruzasvir analysis.

Moving forward, we strongly encourage all HPLC-based dissolution testing to use small internal diameter columns with optimized chromatographic parameters to dramatically reduce the amount of solvent used and amount of waste generated, and that this approach should be implemented at the earliest point possible in drug product development to promote the greatest savings. Additionally, advances in chromatographic instrumentation combined with the techniques outlined here can yield even more benefits with respect to solvent waste reduction for dissolution analyses. For example, the use of dual needle injection systems can greatly reduce the instrument cycle time, which will significantly shorten sequence analysis times and generate less waste than single needle injection systems, this is being investigated in our laboratories. Future advances in software to decouple interfering UV spectra for online fiber optic analyses, the use of dissolution simulations, and software modeling could eventually be used to render offline chromatographic analysis void.

differences are accounted for in the gradient program. The green method developed for this analysis showed a 73% reduction of effluent waste and a 63% reduction in sequence analysis time (Table 2). As can be seen in Figures S2 and S3, the reproducibility, shown as %RSD of the 6 replicates for each time point for each method, and the dissolution profiles compare nearly identically to each other (F2 > 90), confirming the equivalency of the analyses.

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CONCLUSIONS Dissolution is one of the critical analytical tests to monitor release, stability, and guide development of solid drug dosage and is widely used in the life cycle of drug discovery, development, commercialization, and production in the pharmaceutical industry. Analysis of these dissolution samples is most commonly performed by HPLC, which generates significant volumes of aqueous/organic waste, exacerbated by long sequence analysis times. Herein, we describe a simple, fast, and green analytical protocol for dissolution testing based on existing instrumentation. This approach includes utilization of narrow bore HPLC columns, optimization of the chromatographic method, exploitation of system dwell volumes for gradient re-equilibration, and basic chromatographic redevelopment when necessary. Feasibility and reliability are demonstrated through head-to-head comparison of drug release profiles of three marketed combination products analyzed using the USP or filed methods vs our “greener chromatography” methods. Overall, our data demonstrated a roughly 80% reduction of organic/aqueous solution usage and waste generation using optimal method parameters. Furthermore, the analysis run time can be significantly reduced with only a minor impact to that optimized solvent volume reduction, if desired. Finally, implementation only involves replacement of large bore columns with smaller bore or superficially porous particle containing columns, well established column technologies, within existing chromatographic instrumentation. These merits make this approach costeffective, sustainable, and operational with a long lifetime. Given that we estimate more than 10 000 dissolution tests are performed just for our top 5 selling solid dosage drugs per year, adapting this simple and green yet robust and reliable analytical approach into the work flow can have significant reduction of organic solvent and resulting generation of hazardous waste.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04311. Method condition summary for original and proposed “greener” developmental methods investigated, comparison of chromatograms using a 4.6 mm i.d. column on HPLC (filed method) and 2.1 mm i.d. column (DEV1 and DEV 2) on UHPLC systems for JANUMET analyses, comparison of the variability in n = 6 dissolution results at each time point for filed and greened developmental uprifosbuvir (top) and ruzasvir (bottom) chromatography, and comparison of the variability in n = 6 dissolution results at each time point for filed and greened developmental uprifosbuvir (top) and ruzasvir (bottom). (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]; Tel.: +1 215-652-9521. *E-mail: [email protected]; Tel.: +1 215-652-3240. ORCID

Adam Socia: 0000-0003-4754-4948 G


Research Article

ACS Sustainable Chemistry & Engineering Notes

(19) Driver, J.; Raynie, D.; Jackson, P. In A Green Chemistry Assessment for Analytical Methods of Analysis, 13th Green Chem. Eng. Conf., Washington, DC, USA, 2008; American Chemical Society: Washington, DC, USA, 2008. (20) Ng, L. Reviewer Guidance - Validation of Chromatographic Methods. www.fda.gov/downloads/drugs/guidances/ucm134409.pdf. (21) FDA Center for Drug Evaluation and Research (CDER) Guidance for IndustryDissolution Testing of Immediate Release Solid Oral Dosage Forms. www.fda.gov/downloads/drugs/guidances/ ucm070237.pdf (accessed October 06, 2018). (22) Webster, G.; Gragg, M. Scaling LC Methods Using Superficially Porous Particle Stationary Phases. LCGC North Am. 2018, 36 (3), 184−193. (23) Dolan, J. W. Dwell volume revisited. LCGC North Am. 2006, 24 (5), 458 460, 462, 464, 466 .

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Sharon M. O’Brien (Creative Studios of Merck & Co., Inc., Kenilworth, NJ, USA) for helping with the Table of Contents graphic design.

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REFERENCES

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Greener Chromatographic Approaches for Dissolution Testing of Solid

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