44 February / March 2016
which performed adequately in the presence of acid precipitation were typically the ones with the lowest Log D values at pH 1 (Table 2). For example, amphetamine (Figure 14) shows good recovery and has a Log D at pH 1 of -0.8. The extremely high concentration of acidic salts creates a very polar solution and because the amphetamines are also relatively polar at this pH, they are relatively soluble and provide decent recovery. By contrast, EDDP exhibits poor recovery in acid (Figure 18) and displays a high Log D value at low pH implying that it is not soluble in the polar salt solution.
Figure 16: Comparison of the effects of various pretreatment options on Benzoylecgonine. Chromatograms are overlaid with time shift to provide clarity [1].
Analyte
Alprazolam Clonazepam Diazepam
Flunitrazepam Lorazepam Midazolam
Nordiazepam Oxazepam Temazepam Codeine
Hydrocodone
Hydromorphone Morphine 6-MAM
Oxymorphone Phencyclidine
Benzoylecgonine Methadone EDDP
Fentanyl
Norfentanyl Meperidine Naloxone
Norpropoxyphene Propoxyphene Sufentanil Naltrexone
Amphetamine
Methamphetamine MDMA MDA
MDEA Tramadol
Carisoprodol Buprenorphine
Norbuprenorphine
Log P pKa Log D at pH 7 Log D at pH 1 3.02 3.15 3.08 2.55 3.53 3.97 3.21 2.92 2.79 1.34 1.96 1.62 0.9
5 2 3 2
NA 4
2.85 NA NA 9 9 9 9
1.09 0.78 4.49
-0.59 5.01 4.63 3.82 1.42 2.46 1.62 4.52 4.9
3.61 1.36 1.8
2.24 1.86 1.43 2.2
2.45 1.92 3.55 2.3
8.4 8.2 11
9.5 9
9.5 9
10 8 8
10 9 9 9
10 10 10 10 10 9
NA 9
10
3.02 3.15 3.08 2.55 3.69 3.97 3.21 2.92 2.79
-0.52 0.12
-0.11 -1
-1.18 -1
1.35 0.2
2.55 2.18 1.3 -1
1.32 0.43 2.1 2.5 1.8
-0.24 -0.7 -0.5
-1.01 -0.9 -0.5 0.3
1.92 1.51 0.3
Table 2: Comparison of the various Log P values, ionisable pKas and Log D values and specific pH [4].
-1.81 1.17 0.3 0.7
3.69 0.4
0.48 2.92 2.79
-1.84 -1.2
-1.35 -2
-2.44 -2.2 1.35 -0.9 1.89 1.5
0.65
-1.49 -0.66 -1.5 1.74 1.8
0.48 -1.4 -0.8 -0.6 -0.8
-1.24 -0.6 -0.6 1.92 0.9
0.05
Organic Pretreatments 90:10 (v/v) ACN:MeOH
As seen in Figure 15, the opiates responded very well and produced the best results with an osmotic breakdown and ACN/ MeOH precipitation pretreatment step. This information was used as an optimisation step in the ZnSO4
10:90 (v/v) ACN/MeOH
In addition, the majority of the benzodiazepines also produced good response using a combination of ACN and MeOH. However, unlike the opiates, 10:90 (v/v) ACN/MeOH produced more acceptable recovery despite the discoloured supernatant. An example is shown in Figure 17 for the chromatographic overlay of Nordiazepam.
While benzodiazepines carry relatively similar Log P values in comparison to synthetic opiates, their Log D values at pH7 are much different (Table 2) which explains why the majority of benzodiazepines looked best under this pretreatment. It is shown previously [7] that acetonitrile does a poor job extracting hydrophobic analytes in comparison to short chain alcohols. The data here supports this idea that the moderately hydrophobic benzodiazepines require a large amount of a protic solvent like methanol to achieve acceptable solubility or a zinc sulphate modifier that effectively lowers pH [5] enough to reduce the Log D to make the compound soluble in ACN. Lorazepam (Figure 21) is good evidence that ZnSO4
lowers pH as it is seemingly immune to ZnSO4 pretreatment
since it does not contain an ionisable amine group and requires methanol to be extracted.
extraction later described.
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