**1. Introduction**

Thyroid hormones are responsible for the regulation of a variety of metabolic functions, including basal metabolic rate and lipid, glucose and carbohydrate metabolism [1]. This group of compounds contains tyrosine-based compounds including the physiologically active form triiodothyronine (T3) and the prehormone thyroxine (T4). The majority of triiodothyronine is formed enzymatically by the deiodination of thyroxine [2,3] (Figure 1). Hyperthyroidism and hypothyroidism are the two main medical conditions associated with thyroid hormone levels. Hypothyroidism is caused by a depleted level of triiodothyronine, the treatment for which is lifelong thyroid supplement therapy [1,4–7]. Currently, the favoured treatment for hypothyroidism is the administration of levothyroxine sodium salt, with its cost being a considerable factor [8]. However, a number of studies have found that in order to maintain euthyroid levels of both T4 and T3, an excess of levothyroxine sodium salt must be administered [9–11]. This has resulted in products becoming available that contain either T3 alone or a combination of T4 and T3 in a single dose.

Dissolution testing is widely accepted within the pharmaceutical industry as the measure of drug release rate to aid in quality control, formulation and process development [12,13]. Noyes and Whitney, investigating the dissolution of benzoic acid and lead chloride, performed the first reported study into dissolution in 1897 [14]. However, the importance of dissolution testing for pharmaceutical quality control and drug formulation was not established until 70 years later [12,15]. Within the pharmaceutical industry, there are two main categories of dissolution testing performed, biorelevant and quality control [13]. Biorelevant is an abbreviated term for "biologically relevant", and the selected media mimic the fluids found within the stomach (gastric) and intestinal tract [16].

Biorelevant dissolution is a multiple-stage dissolution test designed to model the different in vivo environments as the dosage passes through the gastrointestinal (GI) tract. A biorelevant dissolution method is utilized during early formulation selection and optimization but due to the cost and complexity of the media plus variability of physiological parameters it is replaced by a quality control dissolution method once formulation has been developed [13,15]. A quality control dissolution method

consists of one medium that is designed for detecting variations in routine manufacturing or changes during stability testing (e.g., to detect incorrect granulation or compression [13]).

This paper investigates the bioavailability profile, using simulated in vitro gastrointestinal extraction media, of thyroid hormone supplements that contain either a single or a dual combination of the two hormones using biorelevant dissolution media. In addition, the bioavailability profile is compared against the standard United States Pharmacopoeia (USP) quality control test specification of 70% release within 45 min for individual dosage forms of thyroid hormone supplements [17,18].

**Figure 1.** Conversion of T4 to T3 by deiodination.

### **2. Materials and Methods**

### *2.1. Chemicals and Reagents*

Triiodothyronine and thyroxine/triiodothyronine tablets were purchased from RX Cart (Sagunto, Valencia, Spain), specifically Cytomel containing 25 μg of T3. Tiromel containing 25 μg of T3 and Dithyron containing 12.5 μg of T3 and 50 μg of T4 were purchased from http://www.buyt3.co.uk/. Organic LC–MS grade solvents (methanol, acetonitrile, acetic acid and formic acid) were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Standards of T3 and T4 were purchased from Sigma Aldrich (Poole, Dorset, UK) with a purity of ≥98%. For dissolution media, FaSSIF/FeSSIF/FaSSGF powder was purchased from Biorelevant (London, UK) and additives (sodium hydroxide pellet and monobasic sodium phosphate monohydrate) were purchased from Sigma Aldrich (Poole, Dorset) while sodium chloride, hydrochloric acid and sodium hydroxide were purchased from Fisher Scientific (Loughborough, Leicestershire, UK).
