Chemical analysis of additives included in fully formulated oils using high‐performance liquid chromatography–tandem mass spectrometry

Rationale Fully formulated oils (FFOs) are chemically complex petrochemical products composed of base oil and additive mixtures that are employed in automotive engines to provide lubrication. In particular, the additive portion of FFOs is often precisely controlled to tailor the resultant formulation to a specific role. Analysis of the additive composition of both used and unused FFOs is therefore of great importance within the petroleum, automotive, and wider engineering industries. Methods A simple and rapid reversed‐phase high‐performance liquid chromatography–tandem mass spectrometry method is reported herein for the analysis of a range of additives commonly encountered in FFO samples. Mass spectrometry was performed using an LTQ Orbitrap XL instrument using both positive‐ and negative‐ion electrospray ionization. Tandem mass spectra were acquired in the data‐dependent mode. FFO samples were analysed with minimal sample preparation, limited in this case to simple dilution steps. Results The reported method permits analysis of a range of antioxidant, detergent, and antiwear chemistries from various FFO samples in under 10 min. Additionally, it is demonstrated that additive confirmation can be performed and some structural information obtained using the data‐dependent tandem mass spectrometry method. Furthermore, analysis of additives and corresponding degradation products within a used FFO is reported. Conclusions The results obtained using the reported methodology are of demonstrable use in numerous industries and applications, and readily return an abundance of information on the additive composition of a range of FFO samples. Anticipated applications of the methodology include but are not limited to quality control, suspected counterfeit analysis, and FFO degradation analysis.


| INTRODUCTION
Fully formulated oils (FFOs) play an invaluable role in lubricating mechanical environments such as automotive engines, where they are responsible for increasing efficiency and extending component lifetimes.Typical compositions of FFOs feature hydrocarbon base oil of either mineral or synthetic origin as the major component, into which speciality chemicals are included to modify the resultant formulation for specific applications and extend the effective lifetime of the product.
Within the engine, FFOs can be exposed to high temperatures and entrained gases.In addition, particulate metals such as iron from mechanical surfaces can also be present within the formulation.
[3] To counter this, antioxidant additives are nearly always included in formulations and serve to inhibit oxidative depletion of the FFO. 4,5her commonplace additives, such as antifoaming agents, detergents, dispersants, viscosity modifiers, and zinc dialkyldithiophosphates (ZDDPs), are often included to optimize other chemical and physical properties of the formulation. 6e resultant formulations produced from additives and base oil are very complex, containing many thousands of distinct chemical compounds.[9] Complex mixtures can be challenging to analysts wishing to understand the chemical composition of such samples.10][11][12][13][14][15][16][17] Of these, MS utilizing electrospray ionization (ESI) has found value in the analysis of certain FFO additives. 4,18However, analysis of samples by ESI-MS without prior on-line separation can be hindered by ion suppression, where readilyionizing analytes reduce the ionization efficiency of other analytes and in turn diminish the abundance of the latter species in the obtained mass spectra.Often, reversed-phase high-performance liquid chromatography (HPLC) coupled to MS can help negate ion suppression, whereby components of a complex mixture are separated according to polarity prior to mass spectrometric analysis.
Many modern MS instruments also permit selective dissociation of ions of interest with concomitant mass analysis of the product ions generated in so-called tandem (MS/MS) analyses.These MS/MS analyses are particularly useful for gaining additional structural information for analytes and can allow confident structure assignment of an ion.
Chromatographic methods reported for the analysis of FFO components often target one particular additive class and require 10 min or more to complete. 4,7,10,14,16,17The authors have also reported the development of a dielectric barrier discharge ionization-mass spectrometric methodology for the analysis of selected additives and base oil components of a model FFO. 19 The solvent programme used water and methanol and was as follows: between 0 and 2.5 min, methanol was increased from 75% to 90%, between 2.5 and 2.51 min was then increased to 100% and held until 5.5 min, between 5.5 and 5.51 min the methanol composition was reduced back to 75% and held until 7.5 min.To interface with the mass spectrometer ESI source, the column eluent was split at a ratio of approximately one part to the ESI source and eight parts to waste.
All chromatographic analyses were performed in triplicate.
For mass spectrometric analyses, a Thermo Fisher Scientific LTQ Orbitrap XL with an HESI II source was used.For positive ion electrospray analyses, a source voltage of 3.5 kV was used, with capillary and tube lens voltages set to 30 and 80 V, respectively.For negative-ion electrospray analyses, a source voltage of 3 kV was employed, with capillary and tube lens voltages set to À35 and À110 V, respectively.In both electrospray ion modes, a vaporiser temperature of 250 C and capillary temperature of 275 C was used.
Sheath and auxiliary gas flows were set to 45 and 10 arbitrary units, respectively.An automatic gain control target of 10 6 and maximum inject time of 500 ms were used in both electrospray ion modes with a scan range of m/z 80-2000 in the Orbitrap mass analyser.A lock mass corresponding to N-butylbenzenesulfonamide was used in both positive-and negative-ion electrospray modes.For additional structural information, a data-dependent MS/MS method was employed, where collision-induced dissociation (CID) was used to generate product ions for subsequent ion trap analysis.A range of activation energies between 30 and 50 arbitrary units were used.In some cases where confident assignment of chemical formulae to product ions analysed in the ion trap was difficult, the product ions were instead analysed in the Orbitrap.

| Formulations
To prepare the model FFO formulation in-house, each additive stock solution was prepared at a concentration of 15 mg of the neat additive per millilitre in toluene, with vortexing to ensure complete dissolution.The antifoam additive stock solution was further diluted by a factor of 100 with toluene.For the model formulation, approximately 1 mL was prepared with an overall concentration of 15 mg FFO per 1 mL, according to the formulation outlined in Table 1.For LC-MS/MS analysis, 10 μL of this formulation was made up to 1 mL using toluene and vortexed to ensure thorough mixing.

| Brand samples and used FFO
Selected FFO products available to consumers on the UK market and a used FFO sample were prepared for LC-MS/MS analysis by individually diluting 15 mg of sample in 1 mL of toluene and vortexing.A 10 μL aliquot of the resultant solution was then further diluted to 1 mL using toluene.The properties of the unused FFOs selected for this study are outlined in Table 2.In the case of the used FFO, this was supplied for analysis after use in an engine environment and was of an unspecified mileage.+ Na] + ions is not clear.It is understood these species can be formed from the reaction of ZDDP with alkyl peroxy radicals formed from base oil oxidation within the engine environment, but this pathway is not attributed as the cause for the formation of the disulfide species in the unused and therefore undegraded FFO analysed. 20It is also not proposed that these species are formed during the analytical procedure.In negative-ion electrospray, sulfonate and phenate detergents, as well as DDP ligands of ZDDP complexes, were detected as their corresponding singly charged anions.It is noted that detergents employed in FFOs are manufactured as the metal salt of the corresponding organic acid. 21Given that the authors cannot evidence the nature of the metal counterion, no ion types were specified for the detergents which were detected as their conjugate bases.Identification of the phenolic antioxidant is also possible in negative-ion electrospray from the reporting of [M À H] À ions.
Extracted ion chromatograms (EICs) visualized the MS response of ions of interest throughout the chromatography and were used to profile the elution of analytes of interest.EICs for additives in the model FFO, in both electrospray ion modes, and their chemical identities are shown in Figure 1 and Table 3, respectively.

| HPLC-MS/MS of consumer FFO products of unknown composition
In addition to the analysis of a model FFO, several consumer FFO products of unknown composition were analysed (see Table 2).Aminic antioxidants and ZDDP were found to be present in all formulations, but although all products contained at least one detergent, the exact type of this additive varied between formulations.The Brand B product was found to contain both phenate and salicylate detergents, whereas The ranges for each component are reported at the sponsor's request to protect intellectual property.In practice a precise formulation was used where the percentage amount of each component lies within the range reported in Table 1.
T A B L E 2 Properties of the FFOs used in this study.all Brand A products contained only sulfonate detergents.Also of note is the presence of phenolic antioxidant in some products.Both products 3 and 4, from different brands, contained a well-known phenolic antioxidant employed within lubricant engineering, whereas products 1 and 2, both from Brand A, did not.The differences in composition between products 1-4 are highlighted in Table 4, with EICs reported in Figures S2-S5 of Supporting Information.
Many of the analytes yielded characteristic neutral losses on CID, which allowed confident assignment of additive class.In the instance of detergents, sulfonates readily lose SO 2 with a corresponding decrease in m/z value of 64, whereas salicylate detergents instead lose CO 2 , characterized by a decrease in m/z value of 44. 22For the salicylate detergent with an R group of C 18 H 37 this characteristic loss of CO 2 is particularly useful for distinguishing between the ions derived from a salicylate and phenolic antioxidant of identical exact mass in negative-ion electrospray, which instead undergoes a charge migration fragmentation process leading to the elimination of the observed charged ester moiety, shown in Figure 2.Such charge migration fragmentations are known to occur over conjugated systems. 23P ions also show an abundance of informative product ions, where loss of either a single alkyl group or both alkyl groups and an oxygen to yield the OPS 2 À anion is observed, hence indicating the nature of the alkyl substituents.
In aminic antioxidant dissociation, a complex range of ions related to dissociation along the alkyl chains present on the aromatic rings have been reported in the literature and were observed in this work. 5milar alkyl chain dissociation behaviour is observed for phenate detergents, where a characteristic series of methyl unit losses indicates the substituted group is a saturated linear alkane.A summary of the CID energies which yielded the above characteristic losses are detailed in Figure S6 of the Supporting Information.

| HPLC-MS/MS of used FFO of unknown composition
Across both positive-and negative-ion electrospray analyses, a range of additives were observed within the used FFO sample, in addition to products of degradation.EICs are provided in Figure 3. Principally, a similar variety of undegraded additives was observed when compared to those found in unused products, indicating that in some capacity most additives remain undegraded or are only partially depleted on the timescale of an ordinary oil change interval.In particular, the salicylate and sulfonate detergents were identified within the formulation as their corresponding singly charged anions in negative-ion electrospray and did not appear to have any related products of degradation.Moreover, phenolic and aminic antioxidant species were observed in positive-ion electrospray predominantly as their [M + Na] + and [M + H] + ions, respectively, and were of the same composition as those identified in many of the unused consumer products.Of note is the elution profile of seemingly undegraded DDP species, detected as singly charged anions in negative-ion electrospray, where a second series of elutions was Extracted ion chromatograms of the additive ions detailed in Table 3, representing the elution profiles of additives within the model FFO.DDP, dialkyldithiophosphate; ZDDP, zinc dialkyldithiophosphate. [Color figure can be viewed at wileyonlinelibrary.com] observed after 4.0 min in the chromatography following the initial elution at 0.6 min of largely unretained DDP.Products related to the degradation of the DDP ligands via documented sulphur-oxygen exchange were also observed. 9In the first instance, one sulphuroxygen exchange event yielded a dialkylthiophosphate (DTP) species from DDP on FFO ageing within the engine environment, which may subsequently undergo an additional sulphur-oxygen exchange on continued ageing to form a dialkylphosphate (DP) species.These species were individually observed in these analyses, predominantly as an unretained elution at around 0.6 min, similar to that of the T A B L E 3 Range of ions identified and assignments from the analysis of the model FFO.Ions used for extracted ion chromatograms in Figure 1.
T A B L E 4 Ions identified and assignments from the analysis of a range of commercial products.F I G U R E 2 Dissociation observed for phenolic antioxidant in negative-ion electrospray, leading to a neutral loss of 218 Da and observation of a dominant product ion at m/z 171.This spectrum was acquired in the Orbitrap mass analyser.t-Bu, tert-butyl.
original DDP species from which they are formed.In Figure 3  It is known that certain additives, in addition to ZDDP, are reactive by design within formulations when in use.As such, they are often consumed to form a range of potential degradation products. 7,8Within the used FFO studied, a postulated product of phenolic antioxidant dimerization was assigned the uncharged formula C 50 H 78 O 6 and observed as [M À H] À ions in negative-ion electrospray, yielding a highly intense chromatographic peak at 4.17 min, as shown in Figure 3.This species also formed [M + Na] + ions in positive-ion electrospray, albeit at much lower intensity.
Analysis of fragments generated from CID of the [M À H] À ions were analysed using the Orbitrap mass analyser to provide higher confidence in the chemical formulae and therefore identify the two dominant product ions formed, given in Figure 4.The CID energy used was 30 arbitrary units.The neutral loss of 57 Da in the MS/MS spectrum corresponds to the loss of a butyl radical, in keeping with the presence of known tert-butyl substituents in the undegraded phenolic antioxidant.Furthermore, a loss of 243 Da can be attributed to the formation of an anhydride species on dissociation, a pathway known to occur for phthalate ester-type compounds. 24These assignments are given graphically in Figure 4, with structures that represent a best fit to the measured m/z values and calculated molecular formulae.Diphenylamine-type aminic antioxidants are also known to undergo extensive reactions as the formulation ages on use, but none of these degradation products were identified in the used FFO in this study. 8I G U R E 3 Extracted ion chromatograms of the additive ions detailed in

3 |
RESULTS AND DISCUSSION 3.1 | HPLC-MS/MS of model FFO Chromatographic separation of additives within the model FFO was effective and permitted concomitant mass analysis of many additives of interest within the formulation.In particular, highly polar dialkyldithiophosphate (DDP) ligands from the ZDDP additive were seemingly unretained and eluted first, followed by subsequent elution of antioxidants and detergents.Analyses in positive-ion electrospray predominantly yielded information on antioxidants present within formulations.In particular, an intense response of [M + Na] + phenolic antioxidant ions was observed, with concomitant reporting of lower intensity [M + H] + ions.Additionally, a range of substituted diphenylamine derivatives were chromatographically resolved and identified as [M + H] + ions for the aminic antioxidant additive.Specific species derived from ZDDP additives containing a disulphide moiety were also observed as [M + Na] + ions, but the range of ions observed in positive-ion electrospray for this additive class as a whole was more limited than complementary analyses in negative-ion electrospray.The origin of the disulphide species formed from ZDDP and observed as [M

T A B L E 1
Volumes of additives used to prepare the model FFO. a formulated oil; ZDDP, zinc dialkyldithiophosphate. a they are annotated as DTP and DP degradation products accordingly, and they are denoted in Table5with superscript letter (b).All DDP ions observed throughout these analyses yielded visually identical MS/MS spectra via CID as the DDP species observed in products 1 and 2, suggesting they are chemically highly similar, regardless of retention time.This leads to the conclusion that DDP ions observed after 4.0 min in the chromatography are possibly coordinated to other species in solution as a by-product of FFO ageing within the engine environment, reducing their solvated polarity and hence increasing retention time, and thence within the ESI source prior to mass analysis are released to generate the characteristic singly-charged anions.
+ R 00 + R 000 = C 24 H 52 R group ranges are reported for clarity.Ions were detected with a mass difference of less than 1 mmu.DDP, dialkyldithiophosphate; ZDDP, zinc dialkyldithiophosphate.

Table 5
Selected ions identified and assignments from the analysis of a real-world used FFO.Phenolic antioxidant R = C 7 H 15 to R = C 9 H 19 [M + Na] + 413.3032 a . DDP, dialkyldithiophosphate; DTP, dialkylthiophosphate; DP, dialkylphosphate; ZDDP, zinc dialkyldithiophosphate. [Color figure can be viewed at wileyonlinelibrary.com]T A B L E 5 R group ranges are reported for clarity.Ions were detected with a mass difference of less than 1 mmu.DDP, dialkyldithiophosphate; DP, dialkylphosphate; DTP, dialkylthiophosphate; FFO, fully formulated oil; ZDDP, zinc dialkyldithiophosphate. a Ions used for extracted ion chromatograms in Figure 3. b Ions identified as degradation products.