Figure 9.2 shows an electrospray spectrum of the basic drug acebutolol, the positive charge on the molecule is generated by its basic centre being protonated, this occurs even in a weakly acidic HPLC mobile phase. The main ion seen at m/z 337.2 (m/z stands for mass over charge ratio) is due to the molecular weight of the drug plus a proton. ESI is known as a soft ionisation technique and in Figure 9.2 it can be seen that there is little fragmentation of the molecule. A very minor fragment can be observed at m/z 319.2, which is due to the loss of water (18 amu). Two additional features of the spectrum should be noted:

1. The ion separation used in this case was based on a quadrupole (discussed later) which measures masses to approximately one decimal place and the mass measured for protonated acebutolol (C₁₈H₂₉N₂O₄) is greater than the mass of 337 amu, which would be obtained by adding up all the atoms making it up according to their nominal masses, i.e. C=12, H=1, N=14 and O=16. The greatest deviation from nominal mass is for hydrogen (Table 9.1), which has an exact mass of 1.00783. Hydrogen is very abundant in drug molecules and the presence of 29 hydrogen atoms in protonated acebutolol increases its mass by 29 × 0.00783 = 0.23 above its nominal mass of 337 and thus the quadrupole measures it at approximately 337.2 amu.

2. There is an additional peak which is abundant in the mass spectrum at m/z 338.2. This is due to protonated acebutolol where one of the 18 carbon atoms making up the molecule is due to a ¹³C atom. There is a 1.1% probability that a ¹³C atom will occur and where there are 18 C atoms there is a 1.1×18 = 19.8% probability that one of them will be a ¹³C atom. Thus the ion at m/z 338.2 is approximately 20% of the intensity of the ion at m/z 337.2. The abundances of the isotopes for H, N and O are much lower and do not contribute very much in this case. The halogen atoms chlorine and bromine have abundant isotopes and their isotope peaks can be useful in structure elucidation. The exact masses and isotope abundances of atoms commonly found in drug molecules are summarised in Table 9.1.

Fig. 9.2 A positive electrospray ionisation (ESI) spectrum of acebutolol.

Table 9.1 Elements commonly found in drug molecules with their exact masses, isotopes and isotope abundances

ESI can also produce negative ions if the polarity on the spray needle is reversed so that it attracts positive ions thus producing a negatively charged spray. Figure 9.3 shows the ESI mass spectrum of ketoprofen, which forms a negatively charged anion at m/z 253.1. In addition, the spectrum is dominated by an adduct ion formed between ketoprofen and formate, which was used as an additive in the HPLC mobile phase. Adduct formation can occur both in positive and negative ion mode but is usually more pronounced in negative ion mode. It can occur even with trace levels of contaminants in the mobile phase, and it can be seen that the spectrum of ketoprofen also contains an ion at m/z 289.1, which is due to adduct formation with traces of Cl⁻ in the mobile phase. It can also be observed that there are more background ions present in the spectrum of ketoprofen and the ions at m/z 91.0 and 137.0 are due to clusters of 2 and 3 formic acid molecules. Table 9.2 lists common adduct ions which are observed in ESI spectra.

Fig. 9.3 A negative ion electrospray ionisation (ESI) spectrum of ketoprofen.

Table 9.2 Some of the commonly observed additions to molecular ions observe under electrospray ionisation (ESI) conditions

	Adduct	Comment
Addition + ve ion amu
18	NH₄⁺	More likely to occur with ammonium in the mobile phase
22	Na⁺	Forms readily with traces of Na⁺ in mobile phase
32	CH₃OH	With methanol in mobile phase
39	K⁺	Less common than Na, adduct forms with traces of K⁺ in the mobile phase
41	CH₃CN	Formed with acetonitrile in mobile phase
54	CH₃OH/Na⁺	Formed with methanol in mobile phase + traces of Na⁺
63	CH₃CN/Na⁺	Formed with acetonitrile in mobile phase + traces of Na⁺

Addition −ve ion amu
35	Cl⁻	Formed with traces of Cl⁻ in mobile phase
45	HCOO⁻	With formate in mobile phase
60	CH₃COO⁻	With acetate in mobile phase

Self-test 9.1

Ampicillin forms many adducts. The drug was introduced into the mass spectrometer in a mobile phase containing methanol. Identify the adducts of ampicillin:

Answers: 372 Na, 382 methanol, 446 3 × methanol, 478 4 × methanol, 484 3 × methanol + K.

Atmospheric pressure chemical ionisation (APCI)

Atmospheric pressure chemical ionisation (APCI) is closely related to ESI and instruments carrying out ESI can be readily switched to operate in APCI mode. In APCI mode the eluent from the HPLC does not pass through a charged needle before entering the mass spectrometer source but via a heated tube so that it forms an aerosol. Upon exiting the heated tube an electric discharge is passed through the aerosol generating reactive species such as H₃O⁺ and N₂⁺, which promote the ionisation of the analytes. The technique has never achieved the level of popularity of ESI since most drug molecules can be ionised under ESI conditions; however, APCI can be employed for the analysis of drug molecules of low polarity that do not ionise efficiently under ESI conditions.

Electron impact ionisation (EI)

EI is not compatible with the use of HPLC as a method for introducing the sample into the mass spectrometer. Before ESI was developed several interfaces were developed which were compatible with EI type ionisation, such as particle beam, thermospray and FRIT-EI interfaces, but these have been almost completely superseded by the ESI interface. However, EI is still used in conjunction with sample introduction either via a direct heated probe or via gas chromatography (GC):

(i) The sample is introduced into the instrument source by heating it on the end of a probe until it evaporates, assisted by the high vacuum within the instrument or via a capillary GC column.

(ii) Once in the vapour phase, the analyte is bombarded with the electrons produced by a rhenium or tungsten filament, which are accelerated towards a positive target with an energy of 70 eV (Fig. 9.4). The analyte is introduced between the filament and the target, and the electrons cause ionisation as follows:

Fig. 9.4 Ion generation in an electron impact source.

(iii) Since the electrons used are of much higher energy than the strength of the bonds within the analyte (4–7 eV), extensive fragmentation of the analyte usually occurs.

(iv) The molecule and its fragments are pushed out of the source by a repeller plate which has the same charge as the ions generated.

Figure 9.5 shows the EI spectrum of ketoprofen. Ions generated under EI conditions are always positive and thus the ion at m/z 254 is exactly the molecular weight of ketoprofen minus the very small mass of an electron. If there is an electronegative atom in the molecule, such as oxygen or nitrogen, usually the positive charge is located there. Unlike the ESI spectrum of ketoprofen shown in Figure 9.3 the EI spectrum of ketoprofen contains many fragment ions because of the high energy nature of the ionisation process. This is an advantage when it comes to structural confirmation since the spectrum provides a unique fingerprint of the molecule which can be matched against a library spectrum and also can be interpreted. Identifying all of the fragment ions in an EI spectrum is often not easy and ketoprofen provides a less common example where many of the fragment ions can be explained (Fig. 9.6).

Fig. 9.5 An electron impact spectrum of ketoprofen.

Fig. 9.6 Structure of the ions generated in the electron impact ionisation (EI) mass spectrum of ketoprofen.

Figure 9.7 shows a generalised scheme for decomposition of a molecule under EI conditions. The principles of the scheme are as follows:

(i) M⁺· represents the molecular ion which bears one positive charge since it has lost one electron, and the unpaired electron which results from the loss of one electron is represented by a dot. Such an ion is known as a radical cation.

(ii) M⁺· may lose a radical, which, in a straightforward fragmentation not involving rearrangement, can be produced by the breaking of any single bond in the molecule. The radical removes the unpaired electron from the molecule, leaving behind a cation A⁺.

(iii) This cation can lose any number of neutral fragments (N), such as H₂O or CO₂, but no further radicals.

(iv) The same process can occur in a different order with a neutral fragment (H₂O, CO₂, etc.) being lost to produce B⁺, and since this ion still has an unpaired electron it can lose a radical to produce C⁺; this ion can thereafter only lose neutral fragments.

Fig. 9.7 Electron impact ionisation (EI) fragmentation pathways.

To summarise, the following rules apply to mass spectrometric fragmentation:

(a) The molecular ion can lose only one radical but any number of neutral fragments.

(b) Once a radical has been lost only neutral fragments can be lost thereafter.

Table 9.3 shows typical fragments which are lost under EI conditions to give the complex fingerprint pattern of a particular molecule. Further examples of EI spectra will be discussed later in the chapter.

Table 9.3 Common losses from a molecular ion

Loss amu	Radicals/neutral fragments lost	Interpretation
1	H·	Often a major ion in amines, alcohols and aldehydes
2	H₂
15	CH₃·	Most readily lost from a quaternary carbon
17	OH· or NH₃
18	H₂O	Readily lost from secondary or tertiary alcohols
19/20	F·/HF	Fluorides
28	CO	Ketone or acid
29	C₂H₅·
30	CH₂O	Aromatic methyl ether
31	CH₃O·	Methyl ester/methoxime
31	CH₃NH₂	Secondary amine
32	CH₃OH	Methyl ester
33	H₂O + CH₃·
35/36	CI·/HCI	Chloride
42	CH₂= C=O	Acetate
43	C₃H₇·	Readily lost if isopropyl group present
43	CH₃CO·	Methyl ketone
43	CO + CH₃·
44	CO₂	Ester
45	CO₂H·	Carboxylic acid
46	C₂H₅OH	Ethyl ester
46	CO + H₂O
57	C₄H₉·
59	CH₃CONH₂	Acetamide
60	CH₃COOH	Acetate
73	(CH₃)₃Si·	Trimethylsilyl ether
90	(CH₃)₃SiOH	Trimethylsilyl ether

Matrix assisted laser desorption ionisation (MALDI)

MALDI uses a nitrogen laser to promote ionisation of molecules prior to ion separation in a mass spectrometer. It is usually combined with time of flight (TOF) separation of the ions generated. In order for the sample to be ionised it has to be dissolved in a matrix that absorbs UV radiation at around the wavelength (337 nm) produced by the laser. A simple example of a matrix is dihydroxybenzoic acid and there are a number of similar aromatic compounds which are used to promote ionisation of different classes of molecules. The sample solution is mixed with matrix solution on a metal plate and allowed to dry prior to being introduced into the instrument. The laser is then directed at the target plate to promote ionisation (Fig. 9.8).

Fig. 9.8 Matrix assisted laser desorption ionisation (MALDI) carried out on a sample crystallised with a UV absorbing matrix.

The technique like ESI is a soft ionisation technique and the ions generated are most commonly due to protonated, or in negative ion deprotonated, molecular ions without extensive fragmentation occurring. It has been widely applied in the characterisation of proteins since it allows ionisation of these high-molecular-weight compounds as singly charged ions and, in combination with TOF separation, measurement of high-molecular-weight species can be carried out. It is also a useful technique for the determination of very polar compounds such as DNA oligomers (DNA fragments 10–20 base pairs long) which contain a phosphate backbone and do not readily ionise in ESI mode because of the strong association of these molecules with sodium ions.

MALDI is also useful for the ionisation of other polar biomolecules and Figure 9.9 shows the ions generated from coenzyme A and two acyl CoAs using MALDITOF in negative ion mode. Another use of MALDI in pharmaceutical development is in the characterisation of pharmaceutical polymers. These usually comprise a mixture of chain lengths and MALDIMS can provide a molecular size distribution for these materials providing one method for setting a quality standard for such materials. The disadvantage of MALDIMS is that it is not readily quantitative and cannot be linked directly with HPLC. However, another area where MALDI has proved useful has been in tissue imaging where a tissue section is coated with matrix and the MALDIMS instrument is used to scan across the tissue in ca 0.1 mm steps to image the tissue in terms of the molecules making it up which could for instance lead to a better understanding of a disease process or find the site of accumulation of a drug within the tissue.