Previously reported methods for plant hormone extraction used predominately methanol, methanol mixtures or isopropanol. and comprehensive method for the determination of hormones is usually challenging. == Results == The present work reports a rapid, specific and sensitive method using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) to analyze quantitatively the major hormones found in herb tissues within six moments, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all herb hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of herb material without time-consuming additional steps such as purification, sample drying or re-suspension. == Conclusions == This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study herb developmental processes or herb responses to biotic and abiotic stresses in complex tissues. An example is usually shown in which a hormone profiling is usually obtained from leaves of plants exposed to salt stress in the aromatic herb,Rosmarinus officinalis. Keywords:UPLC/ESI-MS/MS, Phytohormones, Auxins, Abscisic acid, Cytokinins, Gibberellins, Salicylic acid, Jasmonic acid, 1-amino-cyclopropane-1-carboxyic acid,Rosmarinus officinalis == Background == Hormones play a pivotal role in most physiological processes in plants. These structurally diverse compounds ARS-1620 that take action usually at nanomolar levels include five groups of the so-called “classic” hormones, comprising auxins, cytokinins, gibberellins (GA), abscisic acid (ABA) and ethylene, and several other herb growth regulators, including jasmonates, salicylates, brassinosteroids, polyamines or the very recently discovered strigolactones, which fit several of the criteria to be considered hormones [1-3]. Furthermore, the list of herb hormones is usually expected to increase due to a better understanding of herb growth and development and stress responses, and the use of technological improvements in analytical methods. Recent studies support the contention that hormone actions build a signaling network and mutually regulate several signaling and metabolic systems, such as auxins and GAs in growth regulation [4], CKs, auxins, ABA and strigolactones in apical dominance [2,5], auxins and brassinosteroids in cell growth [6,7], ethylene and cytokinins in the inhibition of root and hypocotyl elongation [8], ethylene, ABA and GAs in some herb stress responses [9,10], or SA, JA and auxin in herb responses to pathogens [11,12] to name just a few of the reported hormonal interactions. Therefore, focusing on a single endogenous herb hormone to evaluate hormone-regulated physiological or developmental biological problems is not sufficient anymore [13]. In order to understand better the network regulation of hormone action influencing herb growth and development as well as the distribution of several hormones at the organ, cellular and sub-cellular levels, an ideal analytical method should provide a measure of multiple hormone concentrations (hormonal profiling) ARS-1620 from a single experimental sample. Therefore several methods for the simultaneous quantification of multiple herb hormones LKB1 using mass spectrometry with multiple reaction monitoring (MRM) have been developed recently. It has been reported a multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique for the simultaneous analysis of SA, JA, IAA, ABA and OPDA inArabidopsis thaliana[13]. However, GC-MS is limited to volatile compounds and as a result it is necessary to purify and derivatize hormones prior to analysis. Another potential downside in GC-MS procedures apart from the purification and derivatization is the use of high temperatures, which can degrade thermal labile compounds [14]. An alternative to GC-MS is usually liquid chromatography coupled to mass spectrometry (LC-MS). A high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous analysis of 15 herb hormones and metabolites from four different hormone classes (auxins, cytokinins, GAs and ABA) has been reported to analyze hormone regulation of thermodormancy ARS-1620 of lettuce seeds [15]. Also, a HPLC/ESI-MS/MS method to analyze seven major classes of herb hormones including auxins, cytokinins, GAs, ABA, jasmonates, brassinosteriods and SA inArabidopsis thalianahas been developed [1] . Furthermore, an ultrahigh-performance liquid chromatography.