Simultaneous Quantification of Complex Phospholipid Compositions Containing Monophosphoryl Lipid-A by RP-HPLC

Vorauer-Uhl, Karola; Jeschek, Dominik; Lhota, Gabriele; Wagner, Alexandra; Strobach, Stefanie; Katinger, Hermann

doi:10.1080/10826070903163180

Cited by 9 publications

(7 citation statements)

References 13 publications

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“…Incorporation of MPLA into NLPs was assessed by reverse phase HPLC by modifying published procedures [46]. Briefly, 20 ml concentrated NLP stocks containing MPLA were speed-vacuumed until dry.…”

Section: Quantification Of Mpla Incorporation In Nlpsmentioning

confidence: 99%

The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge

et al. 2013

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Recent studies have demonstrated that therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms; however, the potential of enhancing the efficacy of such treatments using nano-scale delivery platforms requires more in depth evaluation. As such, we employed a nanolipoprotein (NLP) platform to enhance the efficacy of innate immune agonists. Here, we demonstrate that the synthetic Toll-like receptor (TLR) agonists monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG) can be readily incorporated into NLPs. Conjugation of MPLA and CpG to NLPs (MPLA:NLP and CpG:NLP, respectively) significantly enhanced their immunostimulatory profiles both in vitro and in vivo compared to administration of agonists alone, as evidenced by significant increases in cytokine production, cell surface expression of activation markers, and upregulation of immunoregulatory genes. Importantly, enhancement of cytokine production by agonist conjugation to NLPs was also observed in primary human dendritic cells. Furthermore, BALB/c mice pretreated with CpG:NLP constructs survived a lethal influenza challenge whereas pretreatment with CpG alone had no effect on survival.

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Section: Quantification Of Mpla Incorporation In Nlpsmentioning

confidence: 99%

The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge

et al. 2013

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“…Chromatographic separation of complex lipid A samples on reversed phase (RP) columns was followed with ultraviolet–visible spectroscopic, or fluorescence detection, and indeed in these cases, pre‐column derivatization of the sample was needed to attach a chromophore group to the lipid A. Methylated derivatives of lipid A were suitable for the preparative‐scale RP‐HPLC fractionation of lipid A samples using ultraviolet–visible spectroscopic detection at 210 nm . As a detection method for non‐derivatized lipid A, the use of RP‐HPLC coupled with evaporative light‐scattering detector was reported . Up to now, only a few reports have demonstrated the successful application of CE, HPLC or even supercritical fluid chromatography in combination with MS to the analysis of non‐derivatized lipid A.…”

Section: Introductionmentioning

confidence: 99%

“…[38][39][40] As a detection method for non-derivatized lipid A, the use of RP-HPLC coupled with evaporative light-scattering detector was reported. [41][42][43] Up to now, only a few reports have demonstrated the successful application of CE, [44] HPLC [45,46] or even supercritical fluid chromatography [47] in combination with MS to the analysis of non-derivatized lipid A. The major difficulties encountered in the analysis of native lipid A isolates are the amphiphatic character and undefined heterogeneity of lipid A molecules, mainly arising from the many different possible lengths, positions and numbers of acyl chains.…”

Section: Introductionmentioning

confidence: 99%

Characterization of complex, heterogeneous lipid A samples using HPLC–MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution

et al. 2016

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We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.

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“…MPLA has been successfully encapsulated in numerous particulate carrier systems for development of potent vaccine formulations, including emulsions, liposomes and poly (D,L-lactic-co-glycolic acid) nanoparticles. Although many methods are available for the simultaneous quantification of phospholipids and cholesterol, very few methods have been reported for assay of monophosphoryl lipid-A (Vorauer-Uhl et al, 2009). Owing to the nature of lipid A as a bad chromophore, spectrophotometric sensitivity has been enhanced in by derivatization prior to analysis; (Hamdy et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…For MPLA quantification in such formulations, very careful analytical methods must be selected owing to the physical nature of excipients, which prevents the use of many separation techniques. Although many methods are available for the simultaneous quantification of phospholipids and cholesterol, very few methods have been reported for assay of monophosphoryl lipid-A (Vorauer-Uhl et al, 2009). In this context, a sensitive, trustworthy and rapid method for the analysis of lipid A-based adjuvants by HPTLC, which is a commonly used technique for lipid quantification, is presented.…”

Section: Discussionmentioning

confidence: 99%

Development and validation of TLC‐densitometric method for determination of lipid A adjuvant as a bulk and in solid fat nanoemulsions

Minz

Kaurav

Sahu

et al. 2015

Biomedical Chromatography

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A simple, sensitive, selective and precise high-performance thin-layer chromatographic method was developed for determination of lipid A (MPLA) adjuvant as a bulk and in solid fat nanoemulsions. Chromatographic separations were performed on thin-layer chromatography aluminum plates precoated with silica gel 60 F-254 as stationary phase and chloroform-methanol-ethyl acetate solution (10:2:4, v/v/v) as mobile phase. With this solvent system, compact spots for MPLA at Rf value 0.80 ± 0.02 were obtained. Densitometric analysis of MPLA was carried out in absorbance mode at 357 nm. Linear regression analysis for the calibration plots showed good linear relationship with r = 0.9996 in the concentration range of 20-100 ng/spot. The mean values (±SD) of slope and intercept were found to be 7.355 ± 0.006 and 109.52 ± 0.170, respectively. Limits of detection (LOD) and quantitation (LOQ) were observed at 3.096 and 9.382 ng/spot, respectively.The method was validated for precision, accuracy, robustness and recovery as per the International Conference on Harmonization guidelines. Statistical analysis proved that the developed method for quantification of MPLA as a bulk and in solid fat nanoemulsions is reproducible, selective and economical. This method could be applied for quantitative assay of MPLA in lipid-based vaccine formulations.

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Simultaneous Quantification of Complex Phospholipid Compositions Containing Monophosphoryl Lipid-A by RP-HPLC

Cited by 9 publications

References 13 publications

The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge

The use of nanolipoprotein particles to enhance the immunostimulatory properties of innate immune agonists against lethal influenza challenge

Characterization of complex, heterogeneous lipid A samples using HPLC–MS/MS technique I. Overall analysis with respect to acylation, phosphorylation and isobaric distribution

Development and validation of TLC‐densitometric method for determination of lipid A adjuvant as a bulk and in solid fat nanoemulsions

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