PR3103 Pharmaceutical Analysis II

1. PR3103 Pharmaceutical Analysis II Tutorial Question 3

2. Background • Mrs Tong is worried about the leaching of BisphenolA (BPA) from the milk bottle. • You suggest that you can help her analyse the compound in the milk preparation as well as the blood. • From existing protocols in literature, you extracted the following: “…1.5 ml of serum was mixed with 0.5 ml formic acid and vortexed vigorously. Thereafter, sample was spun at 15000 rpm and clear layer subjected to solid phase extraction. The loaded solid phase was washed with water/aqueous buffer before eluting with 4 ml of methanol:dichloromethane (1:1)…”

3. Question • What is the purpose of adding formic acid before extraction? • Formic acid (HCOOH) is used to bring about protein precipitation. • Serum contains interfering proteins that need to be removed prior to extraction. • When the acid is mixed with serum, which has a usual pH of 7.35 - 7.45, this decrease in pH alters the ionisation states of amino acid R groups. • This therefore disrupts the electrostatic interactions that maintain the proteins’ native folded conformations  causes an alteration in their 3-D structures  result in protein precipitation.

4. Question (b) Propose a suitable material for solid phase extraction. Also suggest a suitable pH for the water/buffer used in washing before the elution step. SPE process

5. Structure of BPA Phenolic OH Benzene ring

6. Question (b)Propose a suitable material for solid phase extraction. • Despite the 2 polar phenolic OH groups, BPA has a rather high log P value of 3.3  due to the 2 non-polar benzene rings. • Thus, a suitable ‘lipophilic’ material for reverse phase SPE is that of an aryl-bonded silica gel.

7. Binding between BPA and aryl-bonded silica gel Van de Waals attraction & hydrophobic interactions O Si O O Si O O

8. Question (b) Also suggest a suitable pH for the water/buffer used in washing before the elution step. • The silica gel interacts with BPA via Van der Waals forces and hydrophobic interactions  the water/buffer used to wash the column should not be sufficiently strong to disrupt these interactions. • Hence, the water/buffer should just be mildly acidic  pH 6 - 7.

9. Question (c) It later occurred to you that toxicity of bisphenol A may be attributed to the free form of the compound. Read up about the pharmacokinetics properties of bisphenol A and determine what additional steps (if any) should be introduced to analyze just the free form? • BPA can exist as its free form in the serum, but can also assume 2 additional states by (1) being bound to plasma proteins and/or (2) being chemically modified into metabolite/s. • Hence, so as to analyse just the free form  I should perform a more non-disruptive separation of the proteins and conjugates. • For example, by ultrafiltration  where the larger-sized molecules (i.e. proteins and conjugates) are retained by the filter medium of defined pore sizes, while the free fraction passes through unhindered.

10. Alternative scenario How about a new scenario where total BPA presented in the serum is to be measured instead of just free BPA? • BPA is very significantly bound to plasma proteins (the bound form represents about 90–95% and the free form about 5–10% of the total)  deproteinisation will therefore be an imperative process that must be completed before extraction. • Example: use of formic acid to bring about protein precipitation and subsequent disruption of the compound’s plasma protein binding.

11. Alternative scenario • Next, 2 metabolites of BPA are found to be present as well monoglucuronide conjugate and the monosulfate conjugate. Monosulfate Monoglucuronide • Hence, enzymatic hydrolysis will be needed to convert the metabolites back to BPA. • Glucuronidase enzyme  monoglucuronide conjugate • Sulfatase enzyme monosulfate conjugate

12. Question (d) If you’re trying to detect bisphenol A in feces, what modification(s) do you need to introduce in terms of sample preparation, if any? • Modifications must be made to the sample preparation procedure when trying to detect BPA in faeces. • This is so as faeces are non-liquid in nature, and therefore have a very different consistency from liquid serum. • Hence, an essential modification is to homogenise the sample to reduce its viscosity. • Homogenisation of faeces can be carried out using either: (1) blade homogeniser – a cutting blade is rotated at a suitable speed (rpm) to break up the material; (2) sonicator – an alternating electric current is transformed into mechanical impulses to disrupt the biological sample.

13. Question (d) If you’re trying to detect bisphenol A in feces, what modification(s) do you need to introduce in terms of sample preparation, if any? • Also, as faeces are highly complex and variable in content, many interfering substances including large amounts of protein can confound the analysis of BPA, and should be removed. • Therefore, I would introduce an additional protein digestion step with the use of various proteolytic enzymes (e.g. trypsin, subtilisin, protease I) to enhance sample purification.

14. Thank you! 