Photochemical stability of warfarin potassium in powdered pharmaceutical tablets

Abstract

BACKGROUND:

Warfarin potassium (Wf) commercial tablets originally formulated for adults are ground before administration to pediatric patients and elderly patients with dysphagia.

OBJECTIVE:

The present study investigated the effect of tablet grinding on the photostability of four types of commercial Wf tablets and predicted the photostability of the tablet powders by chemometric analysis.

METHODS:

The photodegradation of Wf content was evaluated by reversed-phase column high-performance liquid chromatography with ultraviolet (HPLC-UV).

RESULTS:

The bulk Wf powder was relatively photostable, whereas ground Wf tablets underwent substantial photodegradation. The photostability of the ground powders of a brand-name Wf commercial tablet and three generic Wf commercial tablets was quantitatively assessed and compared. In certain cases, the Wf in all the three ground generic tablets was less photostable than in the ground brand-name tablets. After 28 days of light irradiation, the Wf content decreased to 69.79% in the brand-name tablets, while it was 31.90% in some generic tablets. To clarify the factors influencing the relative photostability in various Wf formulations, we analyzed the intermolecular interactions between the active ingredient and the excipients by partial least-squares regression analysis based on photostability screening for each additive.

CONCLUSION:

The results suggested that the additives light anhydrous silicic acid and povidone adversely affect the stability of Wf tablets. In addition, the light stability of ground tablets was affected considerably by their formulation.

Keywords

Photostability crushed tablets chemometric analysis warfarin potassium high-performance liquid chromatography-ultraviolet (HPLC-UV)

1. Introduction

Due to the developing metabolic systems, there are many risks in clinical trials for developing pediatric drugs and obtaining regulatory licenses for them [1]. Furthermore, the economic benefit of such drugs is low because of the low demand. These issues prevent the approval of new safe and effective pediatric pharmaceuticals. Therefore, unfortunately, the current situation is that after grinding tablets approved for use by adults [2], the drug dose must be reduced for administration to children. Meanwhile, due to the aging Japanese population, the number of elderly patients who cannot swallow tablets and capsules is increasing. In fact, most pediatric patients cannot swallow tablets either. The most popular method of drug administration to these elderly and pediatric patients who have difficulty swallowing [3] is to grind tablets or capsules, make paper packages containing the ground powder, and administer the ground powder with a fluid meal. Hence, all community pharmacies in Japan grind tablets and/or capsules meant for adult doses and package them in paper cases that are not photoresistant. It is officially recommended that the dispensing pharmacist should help drug administration for patients who have difficulty swallowing or reduce the drug dose. Therefore, information concerning the effect of grinding on tablets and capsules in practical pharmacy was published as a textbook [2]. The textbook provides qualitative information on light resistance and hygroscopicity of more than 7,000 commercial medicines. In contrast, pharmaceutical manufacturers develop dosage forms (meant for adults) that maintain drug quality under various storage conditions, enhance drug gastrointestinal uptake, stabilize drug blood levels, and prolong drug component half-life. Pharmaceutical companies provide information on the chemical stability of official packaged medicines in the supplied dosage form and guarantee their quality. However, they do not provide information on the chemical stability of packed powdered tablets, despite the frequent use of this form dispensed in clinical pharmacies. The lack of such information might be because of the lower risk of photodegradation of solid drugs than that of solutions [4]. Although the number of photosensitive drugs in the market is growing, there is increasing interest in the photodegradation of solid-state medications under common fluorescent lights in rooms [5].

The International Ergonomics Regulatory Harmonization Council (ICH) states that photostability testing is an integral part of the validation of new drugs and formulations [6]. Therefore, the aim of the present study is to establish a novel, efficient, and fast method to screen the effects of various pharmaceutical formulation excipients on the photostability of a photosensitive drug by multivariate analysis [7]. A typical example of a photosensitive drug is warfarin potassium (Wf), and the quality of these tablets is assured by using light-tight packaging. There are also numerous generic versions of Wf in the market, which have been promoted to lower patient medical costs, but there are few reports on the photostability and hygroscopicity of the ground powder of commercial Wf tablets [8,9]. Therefore, the photostability of ground powders of commercial Wf tablets, including generic drugs, was tested in this study. In addition, a novel, quick, and cost-effective method for photostability screening of powder formulations was established based on the high-performance liquid chromatography-ultraviolet method (HPLC-UV) [10–12] and multivariate analysis.

2. Materials and methods

2.1. Materials

Warfarin standard was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Bulk Wf powder (99.9%) was obtained from Eisai Co. Ltd. (Tokyo, Japan). Ground Wf was prepared by grinding bulk Wf powder in a ball mill (Retsch MM200, T/S 18, 20 min; Retsch GmbH, Haan, Germany).

Tablets, each containing 1 mg Wf, were selected as the brand-name drug. Three commercially available generic drugs were also analyzed. The brand-name drug Wf tablets were procured from Eisai Co. Ltd. (a). Warfarin potassium tablets were acquired from Nipro Pharma Corp. (Osaka, Japan) (b); Arefarin^® tablets were purchased from Fuji Pharma Co. Ltd. (Tokyo, Japan) (c); and Warlin^® tablets were obtained from Taiyou Yakuhin Co. Ltd. (distributed by Nihon Generic Co. Ltd., Tokyo, Japan) (d). Specifications for the various Wf tablets are summarized in Table 1. Several lots of four commercial Wf tablets were ground with a ball mill (Retsch MM200) and filtered through a sieve (100–75 μm). The particle size used in the photostability testing did not exceed 100 μm. The additive agents used included lactose monohydrate (lactose monohydrate milled 200M; DMV International, Veghel, The Netherlands), crystalline cellulose (ceolus PH; Asahi Kasei Chemicals Corporation, Tokyo, Japan), d-mannitol and corn starch (Japanese Pharmacopoeia; Fujifilm Wako Pure Chemicals Corporation, Osaka, Japan), magnesium stearate (plant-based; Fujifilm Wako Pure Chemicals Corporation), talc (Fujifilm Wako Pure Chemicals Corporation), light anhydrous silicic acid (Adsolider^®-101; Freund Corporation, Tokyo, Japan), low-substituted hydroxypropyl cellulose (L-HPC^®; LH-11; Shin-Etsu Chemical Co. Ltd., Tokyo, Japan), carmellose (NS-300®; Nichirin Chemical Industries Ltd., Osaka, Japan), carmellose calcium (E.C.G-505^®; Nichirin Chemical Industries Ltd.), hydroxypropyl starch (HPS-101(W); Freund Corporation), hydroxypropyl cellulose (HPC-L; Nippon Soda Co. Ltd., Tokyo, Japan), povidone (Plasdone K-29/32; ISP Technologies Inc., Waterford, MI, USA), and partly pregelatinized starch (PCS PC-10; Asahi Kasei Chemicals Corporation). HPLC-grade propyl-p-hydroxybenzoate standard, acetic acid, acetonitrile, and methanol were purchased from Wako Pure Chemical Industries Ltd. Methyl orange, methyl red, bromocresol purple, bromothymol blue, cresol red, phenolphthalein, and thymolphthalein were commercially available.

Table 1
Warfarin potassium tablet specifications

Product Manufacturer Dosage format Weight (mg) Additive agent

a Warfarin tablets 1 mg Eisai Co., Ltd. Plain (uncoated) tablet 190 Lactose monohydrate, crystalline cellulose, magnesium stearate, low-substituted hydroxypropylcellulose, hydroxypropylcellulose

b WARFARIN POTASSIUM tablets 1 mg Nipro Pharma Corporation Plain (uncoated) tablet 200 Lactose monohydrate, d-mannitol, crystalline cellulose, hydroxypropylstarch, povidone, carmellose, magnesium stearate, light anhydrous silicic acid

c AREFARIN^® tablets 1 mg Fuji Phama Co., Lit. Plain (uncoated) tablet 120 Lactose monohydrate, corn starch, povidone, carmellose calcium, magnesium stearate, talc

d WARLIN^® tablets 1 mg Taiyou Yakuhin Co., Lit. (Distribution source: Nihon Generic Co., Lit.) Plain (uncoated) tablet 230 Lactose monohydrate, light anhydrous silicic acid, magnesium stearate, partly pregelatinized starch, povidone

	Product	Manufacturer	Dosage format	Weight (mg)	Additive agent
a	Warfarin tablets 1 mg	Eisai Co., Ltd.	Plain (uncoated) tablet	190	Lactose monohydrate, crystalline cellulose, magnesium stearate, low-substituted hydroxypropylcellulose, hydroxypropylcellulose
b	WARFARIN POTASSIUM tablets 1 mg	Nipro Pharma Corporation	Plain (uncoated) tablet	200	Lactose monohydrate, d-mannitol, crystalline cellulose, hydroxypropylstarch, povidone, carmellose, magnesium stearate, light anhydrous silicic acid
c	AREFARIN^® tablets 1 mg	Fuji Phama Co., Lit.	Plain (uncoated) tablet	120	Lactose monohydrate, corn starch, povidone, carmellose calcium, magnesium stearate, talc
d	WARLIN^® tablets 1 mg	Taiyou Yakuhin Co., Lit. (Distribution source: Nihon Generic Co., Lit.)	Plain (uncoated) tablet	230	Lactose monohydrate, light anhydrous silicic acid, magnesium stearate, partly pregelatinized starch, povidone

2.2. Warfarin potassium hygroscopicity test

Relative humidity (RH) values were determined against saturated salt solutions of known RH, including lithium chloride (11% RH), potassium acetate (22% RH), magnesium chloride (32% RH), magnesium nitrate (48% RH), potassium iodide (66% RH), copper chloride (70% RH), and sodium chloride (75% RH) [13]. Twenty milligrams Wf was accurately weighed out in a bottle and stored in a controlled-RH desiccator at 40 °C for 1 day. The relative weight change was then determined.

2.3. Warfarin ultraviolet visible (UV-vis) absorption spectra

UV-vis absorption spectra for warfarin were plotted with the Hitachi U-2810 spectrophotometer (Hitachi, Tokyo, Japan). The warfarin standard was diluted with methanol and a mobile phase (water/acetonitrile/acetic acid = 60:40:1) to 10 μg mL⁻¹, and the UV-vis absorption spectra were plotted. The absorption spectrum for one Wf tablet (a) dissolved in 100 mL water was measured. The warfarin and Wf concentrations were ∼9 μg mL⁻¹ and ∼10 μg mL⁻¹, respectively.

2.4. Photostability testing in a 96-well plate with controlled temperature and humidity

The RH values for the sample powders were regulated using a humidity-controlled 96-well plate, as previously reported [14]. A quartz 96-well plate was divided into 32 small compartments, each consisting of three wells. The sample powder and a saturated salt solution were placed in two different wells per compartment, and a small amount of sample was simultaneously measured at different humidity levels. The 96-well plate was sealed with a plain quartz plate and stored at 40 °C. One compartment was at the selected RH. The quartz 96-well plate was used directly in the photostability test. The photostability test was conducted using two cool-white to daylight fluorescent lights (FPL-27EX-N; OHM, Mitsubishi Electric Corporation, Japan) to simulate natural sunlight exposure (4,710–4,260 lx). During irradiation, the 96-well plate slowly rotated on a turntable to ensure uniform sample surface irradiation.

2.4.1. Photostability testing of Wf bulk and ground powder samples

Ten-milligram samples were weighed and placed in one cell of a humidity-controlled 96-well quartz plate set to 48% RH and 75% RH at 40 °C and exposed to fluorescent light. After 0, 1, 3, 7, 14, and 28 days, photo-exposed samples in one cell were dissolved in water and analyzed by HPLC.

2.4.2. Photostability testing of Wf commercial tablets

Commercial Wf tablets were pulverized and passed through a No. 100 sieve screen (<150 μm). Powder samples were weighed (12–18 mg), and each was placed in one cell of a humidity-controlled 96-well quartz plate set to 75% RH at 40 °C and exposed to fluorescent light. After 0, 1, 3, 7, 14, and 28 days, the photo-exposed samples in one cell were dissolved in the mobile phase and analyzed by HPLC.

2.4.3. Photostability screening for Wf mixtures containing various additives

Warfarin potassium and each additive agent (A: lactose hydrate; B: crystalline cellulose; C: d-mannitol; D: corn starch; E: magnesium stearate; F: talc; G: light anhydrous silicic acid; H: low-substituted hydroxypropylcellulose; I: carmellose; J: carmellose calcium; K: hydroxypropyl starch; L: hydroxypropylcellulose; M: povidone; and N: partly pregelatinized starch) were weighed (10 mg; Wf:additive agent = 1:1), mixed well by manual agitation, placed into one cell of a humidity-controlled 96-well quartz plate set to 75% RH at 40 °C, and exposed to fluorescent light. After 0 and 14 days, the photo-exposed sample in a single cell was dissolved in the mobile phase and analyzed by HPLC.

2.5. HPLC analysis

HPLC was conducted on a TOSOH series HPLC system (Tosoh Bioscience LLC, King of Prussia, PA, USA) fitted with a DP-8020 pump (Tosoh), an AS-8021 auto-sampler (Tosoh), and a GL Sciences CN-3 (4.6𝜙 × 25 cm) column (GL Sciences, Fukushima, Japan). All samples were measured at 283 nm wavelength with the UV-8020 variable wavelength detector (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase consisted of water/acetonitrile/acetic acid (60:40:1); the flow rate was 1 mL min⁻¹; and the sample solution volume was 20 μL.

Warfarin standard solutions (final concentrations, 1–10 μg mL⁻¹) were prepared in methanol and diluted with mobile phase consisting of p-hydroxybenzoate (initial concentration, 50 μg mL⁻¹; final concentration, 5 μg mL⁻¹) as an internal standard (IS). The solutions were then analyzed by HPLC under isocratic conditions. A calibration curve was plotted based on the correlation between the peak areas of the warfarin standard/IS and the corresponding warfarin standard concentrations.

2.5.1. HPLC assay of Wf bulk and ground powder samples

After photostability testing, the Wf bulk and ground powder samples in a single cell in a humidity-controlled 96-well plate were dissolved in 20 mL water and sonicated for 15 min. Then, a 0.5-mL aliquot was diluted up to 20 mL with water. Two milliliters of this solution and 1 mL IS were diluted with 10 mL mobile phase to a final Wf concentration of ∼2.5 μg mL⁻¹ and analyzed by HPLC.

2.5.2. HPLC assay of Wf commercial tablets

After photostability testing, powdered Wf tablets in a single cell of a humidity-controlled 96-well plate were dissolved in 10 mL mobile phase [15] and sonicated for 15 min. The solution was filtered through a 0.45-μm PTFE membrane. Then, 4.5 mL filtrate and 0.5 mL IS were mixed (final Wf concentration, ∼6–10 μg mL⁻¹) and analyzed by HPLC.

2.5.3. HPLC assay of photostability screening mixtures of Wf and additive agents

After photostability screening, mixed powder in a single cell of a humidity-controlled 96-well plate was dissolved in 50 mL mobile phase and sonicated for 30 min. The solution was then filtered through a 0.45-μm PTFE membrane. Then, 0.5 mL filtrate and 2 mL IS were diluted to 20 mL with mobile phase to a final Wf concentration of ∼5 μg mL⁻¹ and analyzed by HPLC.

2.6. Surface acid strength determinations for various additive agents

Solid surface acid strength was determined by combining ∼0.1 g additive agent and 5 mL benzene to a test tube and adding three drops of 0.01 M indicator solution. The indicators used were methyl orange (pK_a, +3.5), methyl red (pK_a, +4.8), bromocresol purple (pK_a, +6.3), bromothymol blue (pK_a, +7.1), cresol red (pK_a, +8.0), phenolphthalein (pK_a, +9.3), and thymolphthalein (pK_a, +9.9). Methyl red and phenolphthalein were dissolved in benzene solution. All other indicators were only slightly soluble in benzene and were dissolved in dimethylformamide instead. The acidity and alkalinity of all solid additive agents tested here were easy to determine. The acid strength (H ₀) of an additive agent was derived from the pK_a for two indicators whose colors changed after being added to it [16,17].

Table 2
Warfarin potassium contents following 14-day photostability screening for each additive

Additive agent Manufacturer Day Warfarin potassium content (%) S.D. Welch’s t-test

A Lactose hydrate a, b, c, d 0 100.0 0.20

14 95.53 1.77

B Crystalline cellulose a, b 0 100.0 0.51 **

14 96.40 0.72

C d-mannitol b 0 100.0 0.24 *

14 90.20 1.74

D Corn starch c 0 100.0 0.67

14 94.16 0.24

E Magnesium stearate a, b, c, d 0 100.0 0.48

14 90.66 0.96

F Talc c 0 100.0 1.35 **

14 93.10 1.81

G Light anhydrous silicic acid b, d 0 100.0 0.26 *

14 95.10 1.67

H Low substituted hydroxypropylcellulose a 0 100.0 2.10 *

14 93.51 0.87

I Carmellose b 0 100.0 2.38

14 95.25 1.70

J Carmellose calcium c 0 100.0 1.06

14 94.57 0.28

K Hydroxypropyl starch b 0 100.0 1.37

14 91.50 1.74

L Hydroxypropylcellulose a 0 100.0 1.12

14 88.80 1.79

M Povidone b, c, d 0 100.0 1.92

14 90.81 1.99

N Partly pregelatinized starch d 0 100.0 0.16 **

14 95.73 0.48

	Additive agent	Manufacturer	Day	Warfarin potassium content (%)	S.D.	Welch’s t-test
A	Lactose hydrate	a, b, c, d	0	100.0	0.20
			14	95.53	1.77
B	Crystalline cellulose	a, b	0	100.0	0.51	**
			14	96.40	0.72
C	d-mannitol	b	0	100.0	0.24	*
			14	90.20	1.74
D	Corn starch	c	0	100.0	0.67	**
			14	94.16	0.24
E	Magnesium stearate	a, b, c, d	0	100.0	0.48	**
			14	90.66	0.96
F	Talc	c	0	100.0	1.35	**
			14	93.10	1.81
G	Light anhydrous silicic acid	b, d	0	100.0	0.26	*
			14	95.10	1.67
H	Low substituted hydroxypropylcellulose	a	0	100.0	2.10	*
			14	93.51	0.87
I	Carmellose	b	0	100.0	2.38
			14	95.25	1.70
J	Carmellose calcium	c	0	100.0	1.06	**
			14	94.57	0.28
K	Hydroxypropyl starch	b	0	100.0	1.37	**
			14	91.50	1.74
L	Hydroxypropylcellulose	a	0	100.0	1.12	**
			14	88.80	1.79
M	Povidone	b, c, d	0	100.0	1.92	**
			14	90.81	1.99
N	Partly pregelatinized starch	d	0	100.0	0.16	**
			14	95.73	0.48

(0 day = 100%, n = 3) (Welch’s t-test, *p < 0.05, **p < 0.01).

2.7. Chemometric analysis

To establish a calibration model predicting the effects of additive agents on the commercial Wf tablets, the photostability patterns of the ground commercial tablets were derived from photostability screening results of the mixtures of Wf and each additive agent (A–N; triplicate samples), as shown in Table 2. Wf photodegradation followed first-order reaction kinetics [18–20]. Thus, a semi-logarithmic graph was plotted using Wf content on the ordinate and light irradiation days on the abscissa. These data were obtained in triplicate from the photostability tests on ground warfarin tablets a–d. A highly linear exponential approximation formula for 0–7 days light irradiation was generated by the least-squares method, and its slope was used as the constant photodegradation rate. Twelve photostability patterns for four different ground commercial tablets were used to calculate, calibrate, and validate the best-fit calibration models. The calibration models were calculated using the photostability patterns as explanatory variables and the photodegradation rate constants as objective variables. The best calibration model was determined to minimize the standard error of cross-validation (SECV) caused by the leave-one-out cross-validation method in the partial least-squares regression (PLSR), with the chemometrics software Pirouette v. 4.5 (Infometrix Inc., Bothell, WA, USA) [21–23], and the standard error of calibration (SEC) (Eq. ((1))). The correlation coefficient constants (r) for the calibration and validation were evaluated. $\begin{eqnarray} \displaystyle \mathit{SEC}=\sqrt{\frac{\mathit{PRESS }}{n_{v}-k}} & & \displaystyle \end{eqnarray}$ (1) where PRESS is the prediction residual error sum of squares, n _v is the validation sample, and k is the number of factors in the model.

3. Results and discussion

3.1. Hygroscopicity of Wf

The relationship between the amount of moisture absorbed and the relative humidity of the Wf bulk powder after 1 day are shown in Fig. 1. Wf stored at 75% RH and at 70% RH deliquesced after 1 day. At 66% RH, a portion of the Wf had deliquesced. Above 66% RH, the weight of Wf had significantly increased. In contrast, the powder remained slightly solidified after storage at 48% RH and 32% RH, and the powder weight increased only slightly. As Wf is highly hygroscopic, humidity control is crucial during the evaluation of its photostability.

Fig. 1.

Hygroscopicity of warfarin potassium after 1 day (*: full deliquescence; **: partial deliquescence).

3.2. UV-vis absorption spectra of warfarin

Figure 2 shows that the UV-visible absorption spectra for warfarin standard and Wf tablet (a) distinctly differed. The UV spectrum for the aqueous tablet (a) solution had one peak at ∼310 nm, but the spectrum for the mobile solvent phase of the warfarin standard had two peaks, at 283 nm and 305 nm. When an acidic mobile phase was used as the solvent, the tablet (a) spectrum shifted to a shorter wavelength and had the same spectral shape as that of the warfarin standard [24]. Wf spectra vary with pH [25]. Hence, care must be taken when quantifying this material by UV-vis absorption spectrophotometry. As all HPLC samples are diluted with the mobile phase, it is expected that the spectral shapes for both warfarin standard and Wf would be the same and could be compared at the same wavelength in HPLC.

Fig. 2.

UV-visible absorption spectra of warfarin standard and warfarin potassium tablet (a).

3.3. Photostability tests

3.3.1. Wf bulk and ground powder samples

Before the photostability testing, the crystal structure and crystallinity of the bulk and ground Wf powder were confirmed to be the same by X-ray powder diffraction. In the photostability tests on the intact Wf bulk powder and ground powder samples in the 96-well plates, the relative changes in the appearance of both samples were investigated. At 48% RH, both powders were virtually unchanged for 7 days. After 14 days, however, their color had changed from white to pale yellow. In contrast, at 75% RH, the white Wf powder quickly turned liquid, and its color turned yellow-brown after 14 days of irradiation. There was no difference between the bulk and ground Wf powders in their crystalline form and their powder appearance. Therefore, the Wf content of the unirradiated (0-day) samples was assumed to be 100%. The sample powders were illuminated for different durations (1, 3, 7, 14, and 28 days), and their drug contents were analyzed by HPLC with a UV detector.

Figure 3 shows the semi-logarithmic plots of the photodegradation for the Wf content in the bulk powder and the ground Wf at 75% RH and at 48% RH. The data are expressed as means ± standard deviation (SD). The mean recoveries were 99.79–100.17% (0 day) and 92.64–98.65% (28 days), and the SDs were in the range of 0.52–2.56%. The intact bulk powder and the ground Wf did not differ in terms of warfarin content even after 28 days under fluorescent irradiation. However, they became liquid at 75% RH. At 48% RH, the intact bulk powder and the ground product did not differ in terms of warfarin content. The Wf content was evaluated by Welch’s t-test. There was no significant difference between the intact bulk powder and the ground product after 28 days of light irradiation at 75% RH or 48% RH. There were also no significant differences between the intact bulk powder at 75% RH and that at 48% RH or between the ground product at 75% RH and that at 48% RH. Therefore, grinding the intact bulk powder did not alter its photostability. It was confirmed that the intact Wf and the ground Wf each showed a straight line on the semi-logarithmic plot and followed the photolysis reaction following the apparent first-order reaction. However, the reaction rate constants at each humidity level did not show a significant difference between the uncrushed product and the crushed product.

Fig. 3.

Time course of photodegradation of warfarin potassium content in bulk powder and ground warfarin potassium (mean ± S.D., n = 3).

3.3.2. Ground Wf commercial tablets

The photostability of the ground powders of the brand-name Wf tablets (a) and three different generic drugs (b–d) were measured. The Wf content of the unirradiated (0-day sample) commercial Wf tablets was assumed to be 100%. The samples were illuminated for different durations (1, 3, 7, 14, and 28 days) and analyzed by HPLC with a UV detector. Figure 4 shows the semi-logarithmic plots of Wf photodegradation in ground tablets (a–d). Data are represented as means ± SD. The photostability tests on the commercial Wf tablets disclosed no changes in appearance. Nevertheless, HPLC revealed a change in warfarin content. The Wf content of the brand-name tablet a had decreased to 95.75%, 90.35%, 87.51%, and 69.79% after 3, 7, 14, and 28 days of irradiation, respectively. In contrast, the three generic tablets were less photostable than the brand-name tablet was. The Wf content of tablets b and c decreased by ∼10% after 3 days. After 28 days, the Wf content had declined to 49.73% and 44.82% for tablets b and c, respectively. Moreover, tablet d demonstrated the largest decrease in Wf content of all four samples. The Wf content for tablet d was 82.07%, 67.18%, 58.44%, and 31.90% after 3, 7, 14, and 28 days of irradiation, respectively. The photostability of the commercial Wf tablets was substantially lower than that of the ground bulk powder products subjected to the same conditions. Therefore, the drug content of the ground tablets during the photostability test was considerably affected by interactions of the active ingredient with the additive agents. All the photodegradation profiles of the commercial Wf tablets had sufficient straight lines with different slopes on the semi-logarithmic plots, meaning that the reaction followed apparent first-order kinetics, including interaction with different additives.

Fig. 4.

Time course of photodegradation of warfarin potassium tablets (mean ± S.D., n = 3).

Figure 5 shows the drug content profile of ground commercial Wf tablets irradiated by fluorescent light for 28 days. The drug content of the bulk powder declined to 92.48%, whereas that of tablets a, b, c, and d had fallen to 69.79%, 49.73%, 44.82%, and 31.90%, respectively. Considering the drug content of the unirradiated tablets as 100%, relative differences among the photostabilities of commercial Wf tablets were identified by Tukey’s test. The ratios of the remaining Wf content in the brand-name tablet a significantly differed from those for the three generic drugs (P < 0.01). Moreover, there were significant differences between tablets c and d and between tablets b and d (P < 0.01).

Fig. 5.

Variation in warfarin potassium content among warfarin potassium tablets after 28 days (mean ± S.D.) (Tukey test; **p < 0.01).

3.3.3. Chemical interactions between Wf and each additive in the ground tablets

The foregoing results suggest that the additive agents may influence warfarin photostability. Hence, the effects of each additive agent on the photostability of Wf were separately examined. The Wf levels after 14-day photostability screening in the presence of each additive are shown in Table 2. Wf with all additives except alpha starch (N) showed a color change. Wf combined with lactose hydrate (A), crystalline cellulose (B), and d-mannitol (C) changed from white to brownish-yellow. Wf combined with corn starch (D), talc (F), light anhydrous silicic acid (G), low substituted hydroxypropyl cellulose (H), carmellose (I), carmellose calcium (J), and hydroxypropyl starch (K) went from white to pale yellowish. Wf combined with hydroxypropyl cellulose (L) became yellowish. Wf combined with magnesium stearate (E) turned brown. Wf combined with povidone (M) changed from a white powder to a yellow-brown oil. In all cases, including those presenting with no color change, the Wf content decreased to 3.5–10.8% of the initial level at 14 days after light irradiation. Welch’s t-test demonstrated no significant difference between (A) and (I) before and after light irradiation but did show significant differences among the other additives (P < 0.01 for B, D, E, F, J, K, L, M, and N; P < 0.05 for C, G, and H). Although the effects of the individual additives on Wf photodegradation could be statistically discerned, as shown in Table 3, the complex interaction between multiple additives and Wf photodegradation could not be explained.

Table 3
Acid strength of each additive agent

Additive agent H ₀

A +4.8 − +6.3

B +4.8 − +6.3

C +4.8 − +6.3

D +4.8 − +6.3

E +6.4 − +7.1

F +3.4 − +4.8

G +3.4 − +4.8

H +4.8 − +6.3

I +4.8 − +6.3

J +4.8 − +6.3

K +4.8 − +6.3

L +4.8 − +6.3

M +4.8 − +6.3

N +4.8 − +6.3

Warfarin potassium +9.3 − +9.9

Additive agent	H ₀
A	+4.8 − +6.3
B	+4.8 − +6.3
C	+4.8 − +6.3
D	+4.8 − +6.3
E	+6.4 − +7.1
F	+3.4 − +4.8
G	+3.4 − +4.8
H	+4.8 − +6.3
I	+4.8 − +6.3
J	+4.8 − +6.3
K	+4.8 − +6.3
L	+4.8 − +6.3
M	+4.8 − +6.3
N	+4.8 − +6.3
Warfarin potassium	+9.3 − +9.9

3.4. Effects of pH of the additive agents on the photodegradation of Wf

Since selection of the pH of the additives is one of the important factors to prevent the degradation of the main drug in pharmaceutical formulations during storage, the pH values of all additives were measured at room temperature (20–25 °C) and ordinary RH. The pH range of the bulk Wf powder in aqueous solution was 7.2–8.3 according to the insert of the tablet package (a). The pH of the solid bulk Wf powder indicated a basicity in the range of 9.3–9.9. Table 3 shows the acid strength of each additive agent. H ₀ decreases with increasing acidity. Most additives were only weakly acidic. (F) and (G) were slightly more acidic, and (E) was closer to neutrality than the other additives. As Wf is alkaline, acidic additives such as (F) and (G) might accelerate its decomposition. However, no relationship was found between additive acidity and decrease in Wf content under irradiation. The acidity levels of the solid surfaces of (F) or (G) might have been too weak to induce Wf decomposition. The effects of each additive on Wf could not be determined here as it was first necessary to consider the relationship between the amount of additive and Wf. Nevertheless, the additives markedly influenced the outcomes of the photostability tests. Additive types and quantities vary greatly among drug manufacturers. Thus, Wf photostability may vary among tablets containing different additives.

3.5. Chemometric analysis of interactions between Wf and additives in tablet formulations

To bring out the efficacy of pharmaceutical preparations safely and efficiently in the human body, the preparations comprise a main bulk drug with pharmacological effects and many pharmaceutical additives, and those components are spatially arranged three-dimensionally and prepared by various pharmaceutical technological methods. Therefore, it is not easy to analyze and predict these complex interactions from the individual characteristics of the components in pharmaceutical preparations. In this study, therefore, the predictive method for the photostability of Wf in the pharmaceutical formulation with complex additives was determined based on the photodegradation properties of the individual additives in the formulation obtained in the above section by chemometric techniques. Photodegradation patterns of the commercial Wf tablets depended on the photosensitivity of all the additives, as indicated in Table 3, and these patterns represented Wf photodegradation characteristics of the complex formulation in the commercial tablets.

Figure 6 shows the photodegradation patterns for ground commercial tablet samples. To predict the photodegradation rate of the commercial pharmaceutical preparation consisting of complex formulations, PLS calibration models were established based on the photodegradation patterns (Fig. 6) of Wf with different additives by the leave-one-out cross-validation method in the PLS.

Fig. 6.

Photodegradation patterns of ground commercial tablet samples used for calibration and validation.

The chemometric parameters for the best-fit PLS calibration model were as follows: number of latent variables (LV) = 4; SECV = 3.71⁻³; SEC = 2.75⁻³; PRESS Val = 6.10⁻⁵; and PRESS Cal = 1.65⁻⁴. The r-values of calibration and validation data were 0.968 and 0.988, respectively. The relationships between the predicted and measured values are shown in Fig. 7 and are represented approximately as straight lines, respectively. $\begin{eqnarray}\displaystyle & \text{} & \displaystyle \text{}\text{Validation result:}∼y=0.956x-0.0019\end{eqnarray}$ (2) $\begin{eqnarray}\displaystyle & \text{} & \displaystyle \text{}\text{Calibration result:}∼y=0.975x-0.0009.\end{eqnarray}$ (3)

The plots of the calibration and the validation had sufficient linearity. Their coefficients of determination (R ²) were 0.977 and 0.938, respectively, and their slopes approached unity.

Fig. 7.

Relationships between predicted and measured values of best-fit calibration models to predict the slope of the first-order reaction rate constant.

To clarify the molecular interaction between Wf and its pharmaceutical additives in photodegradation, the regression vector (RV) of the best fitted PLS calibration models based on Fig. 7 was investigated. The RV of the PLS calibration model is known to statistically provide scientific evidence for the increase or decrease in the predicted value in the calibration model [21,22]. Figure 8 shows the RV of the PLS calibration models used to predict the Wf content in ground powder of the commercial Wf tablets. There were significant positive peaks at light anhydrous silicic acid (G) and povidone (M) but significant negative peaks at d-mannitol (C) and hydroxypropylcellulose (L). Therefore, the result of the RV statistically indicated that the additive agents (G) and (M) had negative impacts on Wf stability, whereas the additive agents (C) and (L) positively affected Wf stability. The RV result suggested that the additive (G) in tablets b and d and additive (M) in tablets b, c, and d were largely responsible for Wf decomposition. The chemometric analysis suggested that the stability of each tablet might be predicted based on the database of Wf photodegradation by individual additives.

Fig. 8.

Regression vectors of best-fit calibration model to predict warfarin potassium content in response to combination with additive agents (A–N).

4. Conclusion

Photostability tests in humidity-controlled 96-well plates could effectively and rapidly evaluate Wf compounds under various levels of atmospheric humidity. Wf bulk powder was highly hygroscopic and fully deliquesced at 66% RH. Nevertheless, this hygroscopic response had virtually no impact on Wf photostability. Furthermore, Wf bulk powder remained photostable across various light exposure durations at various humidity conditions. In contrast, the ground commercial Wf tablets were relatively less photostable in the long term than pure ground Wf powder. In the photostability test of the ground powder, the brand-name tablet a had a higher photostability than that of generic tablets. This discrepancy in the photostability of commercial Wf tablets might ascribed to the differences in the excipients in the brand-name and generic Wf tablets. A chemometric analysis based on the results of photostability tests on each excipient and the Wf itself indicated that light anhydrous silicic acid (G) and povidone (M) had the strongest negative effect on Wf stability. Thus, the chemometric method could be effectively applied to analyze photodegradation reactions of the ground powder samples of commercial Wf tablets containing complex formulations based on the photodecomposition properties of individual pharmaceutical additives.

Footnotes

Acknowledgements

This research received financial support in part from Musashino University Creating Happiness Incubation.

Conflict of interest

The authors declare that they have no conflicts of interest to disclose.

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