Model 2996 pda detector

Vitamin C content was analyzed as previously described^{35 (link)}. Frozen samples (3 g) were ground to a fine powder in liquid nitrogen, extracted twice with 10 ml 1.0% (w/v) oxalic acid and centrifuged at 5000 rpm for 5 min. Each sample was filtered through a 0.45-µm cellulose acetate filter. HPLC analysis of vitamin C was conducted using a Waters instrument with a Model 2996 PDA detector (Waters Inc., Milford, USA). Samples (20 µl) were separated at room temperature on a Waters Spherisorb C18 column (250 × 4.6 mm id; 5 µm particle size) using a 0.1% oxalic acid solvent at a flow rate of 1.0 ml min⁻¹. The amount of ascorbic acid was calculated from absorbance values at 243 nm, using authentic ascorbic acid as a standard. Results were expressed as mg 100 g⁻¹ FW.

Aliquots of microparticles equivalent to 25 mg HCTZ and 12.5 mg CPT were weighed into a 100 mL volumetric flask using a Mettler^® Toledo AG135 balance (Mettler Instruments, Zurich, Switzerland). The samples were made up to volume with 50 mL MeOH and 50 mL 0.1 M HCl and sonicated for 2 h using an ultrasonic bath (Ultrasonic Manufacturing Company, Krugersdorp, South Africa). A 10 mL aliquot of the solution was filtered using a 0.45 μm HVLP filter (Millipore Co., Bedford, MA, USA), then 1 mL of the filtrate was transferred to a 10 mL A Grade volumetric and made up to volume with HPLC grade water. The sample was analysed using a previously validated reversed phase high-performance liquid chromatographic (RP-HPLC) method [23 ]. The RP-HPLC system was comprised of a Waters^® Alliance Model 2695 separation module and a model 2996 PDA detector (Waters^®, Milford, MA, USA) set at 210 nm and separation was achieved using a 5 μm BDS Hypersil^TM C₁₈ 250 mm × 4.6 mm i.d. column (Thermo Fischer Scientific Inc., Johannesburg, South Africa) using a mobile phase of methanol: water in a 40:60 v/v ratio adjusted to pH 3.0 using 85% v/v orthophosphoric acid [23 ]. The encapsulation efficiency (% EE) was calculated using Equation (1).
$\% EE=\frac{\mathrm{actual}\mathrm{API}\mathrm{loaded}}{\mathrm{theoretical}\mathrm{API}\mathrm{loaded}}\times 100$