*3.2. Reagents and Solutions*

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#### *3.2. Reagents and Solutions*

Memantine (MEM), Amantadine (AMA) and Rimantadine (RIM) were purchased by Sigma; *o*-phthalaldehyde (OPA, Fluka), *N*-acetylcysteine (NAC, Merck), Na2CO3 (Merck), NaOH (Merck) and HCl (Sigma) were all of analytical grade. Doubly de-ionized water was produced by a Milli-Q system (Millipore).

The stock solutions of the analytes were prepared at the 1000 mg L−<sup>1</sup> level in water. They were kept refrigerated and were found to be stable for at least one month. Working solutions were prepared on a daily basis by appropriate dilutions in water. The 100% level was 100 mg L−<sup>1</sup> for MEM and AMA and 10 mg L−<sup>1</sup> for RIM.

The derivatizing reagen<sup>t</sup> (OPA) was prepared at an amount concentration of 10 mmol L−<sup>1</sup> by firstly dissolving in 0.5 mL methanol and subsequently adding 9.5 mL water [19]. This solution was stable for two weeks at 4 oC in an aluminum foil-wrapped container. NAC was prepared in water (10 mmol <sup>L</sup>−1) and diluted at the desired working concentrations in 100 mmol L−<sup>1</sup> carbonate bu ffer (pH = 10).

The placebo mixture used in accuracy and selectivity studies consisted of representative pharmaceutical grade excipients and was prepared at a nominal concentration of 10 mg mL−<sup>1</sup> according to previously published protocols [20].

#### *3.3. ZF Procedure*

A graphical depiction of the ZF sequence can be found in Figure 3. In brief, NAC/Bu ffer (75 μL, 2.5 mmol <sup>L</sup>−1, pH = 10), OPA (50 μL, 5 mmol <sup>L</sup>−1) and sample (100 μL) were sequentially aspirated in the holding coil (HC). The reaction mixture was propelled for 30 s towards the reaction coil (RC) at a flow rate of 0.6 mL min−1, and the reaction was allowed to develop for 60 s under stopped-flow conditions. Detection was carried out downstream at λex/λem = 340/455 nm. More detailed description of the ZF steps is tabulated in Table S1 (supplementary material). The sampling throughput was 16 h−1.

#### *3.4. Preparation of Samples*

Pharmaceutical samples included both EU-licensed products—purchased from local pharmacies—and non-EU-licensed formulations—purchased from web-pharmacies. Three lots of MEM oral drops (10 mg mL−1) were analyzed directly, after 10-fold dilution in water. AMA capsules (100 mg per cap) and MEM tablets (5 mg per tab) (*n* = 10) were ultrasonically dissolved in water followed by filtration through 0.45 μm syringe filters and dilution with water to meet the 100% level concentration. For content uniformity tests, ten capsules or tablets from each formulation were treated individually (*n* = 10) as indicated by the U.S. pharmacopoeia [21].

#### *3.5. HPLC-PCD Corroborative Method*

Fifty microliters of samples or standards were injected in HPLC column and separated at ambient temperature at a flow rate of 0.5 mL min−1. The HPLC mobile phase was CH3OH/phosphate bu ffer (25 mmol <sup>L</sup>−1, pH = 2.0) at a volume ratio of 50:50. Prior to use, it was filtered under vacuum through 0.22 μm membrane filters (Whatman ®). The post-column-derivatization reagents were OPA at an amount concentration of 20 mmol L−<sup>1</sup> and a mixture of NAC (5 mmol <sup>L</sup>−1)/<sup>100</sup> mmol L−<sup>1</sup> borate bu ffer (pH = 11.0). The PCD reagents were pre-mixed on-line (0.2 mL min−<sup>1</sup> each) through a binary inlet static mixer (BISM, ASI-Analytical Scientific Instruments) with internal volume of 250 μL. The derivatization was allowed to proceed on passage through a 100 cm long knitted reaction coil. The derivatives of Amantadine, (*<sup>t</sup>*R = 6.6 min), Rimantadine (*<sup>t</sup>*R = 13.5 min), Memantine (*<sup>t</sup>*R = 15.8 min) were detected fluorimetrically at λex/λem = 340/455 nm (see typical chromatogram in Figure S3 in the supplementary material).

#### **4. Conclusions**

The developed automated fluorimetric sensor for the analysis of pharmaceutically active adamantane derivatives offers some interesting features:


**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/21/3975/s1, Figure S1: Schematic diagram of the HPLC-PCD setup; Figure S2: Reaction between OPA and the studied adamantine derivatives using N-acetylcysteine (NAC) as nucleophilic reagent, Figure S3: Representative chromatogram from standard mixture of the adamantane derivatives by the corroborative HPLC-PCD method, Table S1: ZF sequence for the automated determination of adamantane derivatives.

**Author Contributions:** Conceptualization, P.D.T. and C.K.Z.; methodology, P.D.T. and C.K.Z.; validation, P.D.T., S.P. and C.K.Z.; data curation, P.D.T., S.P. and C.K.Z; writing—original draft preparation, P.D.T. and C.K.Z.; writing—review and editing, P.D.T. and C.K.Z.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
